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FATE OF VIRUSES FOLLOWING SEWAGE SLUDGE 
APPLICATION TO SOILS 



By 

OSCAR CARLOS PANCORBO 



A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF 

THE UNIVERSITY OF FLORIDA 

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE 

DEGREE OF DOCTOR OF PHILOSOPHY 



UNIVERSITY OF FLORIDA 
1982 



This dissertation is dedicated to my parents, 
and to Ambrosina, Adrianne and Amanda. 



ACKNOWLEDGEMENTS 

The author would like to acknowledge and sincerely thank the 
chairman of his doctoral committee, Dr. Gabriel Bitton, for his wisdom, 
patience and encouragement during the course of this study, and for his 
assistance in developing this dissertation. The author is also grateful 
to the other members of the committee. Dr. Thomas L. Crisman, Dr. Dale 
A. Lundgren, Dr. George E. Gifford, Dr. Samuel R. Farrah, and Dr. Allen 
R. Overman, for the advice and guidance they extended to him. 

The author also expresses his thanks to other faculty members, 
including Dr. James M. Davidson and Dr. John Cornell, for their advice 
on various phases of this study. 

The author is indebted to Mr. Orlando Lanni for his excellent 
technical assistance. 

The assistance of Mr. Albert White, Kanapaha wastewater treatment 
plant. City of Gainesville, Florida, is acknowledged. The author also 
wishes to thank Dr. Gerald H. Elkan, Department of Microbiology, North 
Carolina State University, Raleigh, for the loan of the hydrostatic 
pressure chamber. 

Special thanks are extended to fellow students, in particular, Mr. 
Phillip R. Scheuerman, for their insights during the course of this study. 

This work was supported by grant No. R804570 from the United States 
Environmental Protection Agency. 

Finally, the author acknowledges with gratitude his wife, Ambrosina, 
for her understanding and patience during his graduate study. 

i i i 



TABLE OF CONTENTS 



Page 

ACKNOWLEDGEMENTS i i i 

ABSTRACT vii 

CHAPTER 

I INTRODUCTION 1 

II LITERATURE REVIEW 4. 

Viral Pathogens Found in Raw Wastewater 4 

Removal of Viruses by Wastewater Treatment Processes 5 

Primary Sedimentation 5 

Activated Sludge 13 

Removal of Viruses by Sludge Treatment Processes 16 

Viruses in Raw Sludges 16 

Sludge Treatment Processes 18 

Viral and Other Health Hazards Associated 

with Treated Sludges 42 

Final Sludge Disposal 43 

Fate of Sludge-Associated Viruses in Soils 49 

rxr EFFECT OF SLUDGE TYPE ON POLIOVIRUS ASSOCIATION 

WITH AND RECOVERY FROM SLUDGE SOLIDS 50 

Introduction 50 

Materials and Methods 52 

Virus and Viral Assays 52 

Sludges 56 

Association of Seeded Poliovirus 

with Sludge Solids 59 

Recovery of Seeded Poliovirus from 

Sludge Components 65 

Statistical Treatment of Data 67 

Results and Discussion 67 

Association of Seeded Poliovirus 

with Sludge Solids 67 

Recovery of Solids-Associated Viruses 75 



TV 



CHAPTER 



Page 



IV POLIOVIRUS TRANSPORT STUDIES INVOLVING SOIL 
CORES TREATED WITH VIRUS-SEEDED SLUDGE UNDER 

LABORATORY CONDITIONS 82 

Introduction 82 

Materials and Methods 83 

Virus and Viral Assays 83 

Primary Wastewater Effluent 84 

Sludges 84 

Association of Seeded Poliovirus 

with Sludge Solids 91 

Rain Water 92 

Soils 94 

Poliovirus Transport Studies 94 

Effect of Soil Bulk Density 

on Poliovirus Transport 108 

Results and Discussion 116 

Poliovirus Suspended in 0.01 N CaClo 117 

Poliovirus Suspended in Diluted 

Anaerobically Digested Sludge 120 

Poliovirus Suspended in Undiluted 

Anaerobically Digested Sludge 150 

Condi tioned-Dewatered Sludge 158 

Chemical Sludges 161 

Lime-Stabilized, Chemical Sludges 170 

1/ RETENTION AND INACTIVATION OF ENTEROVIRUSES 

IN SOIL CORES TREATED WITH VIRUS-SEEDED SLUDGE 

AND EXPOSED TO THE NORTH-CENTRAL FLORIDA ENVIRONMENT 178 

Introduction 173 

Materials and Methods I79 

Viruses and Viral Assays I79 

Sludges I81 

Association of Seeded Viruses 

with Sludge Solids 181 

Soil 1g2 

Fate of Viruses in Soil Cores 183 

Virus Recovery Procedures 190 

Measurement of Environmental Parameters I93 



CHAPTER Page 

Results and Discussion 196 

Association between Seeded Enteroviruses 

and Sludge Solids 197 

"First Survival Experiment (7 October 

1977-12 October 1977) - — 197 

Second Survival Experiment (2 June 

1978-24 August 1978) 200 

Third Survival Experiment (11 October 

1978-20 January 1979) — 211 

VI MONITORING OF INDIGENOUS^ ENTEROVIRUSES AT TWO 

SLUDGE DISPOSAL SITES IN FLORIDA 222 

Introduction 222 

Materials and Methods 224 

Sludge Disposal Sites 224 

Virus Recovery Procedures 236 

Viral Assays 242 

Weather Data 242 

Results and Discussion 244 

Kanapaha Sludge Disposal Site — 244 

Jay Sludge Disposal Site 244 

VII EFFECT OF HYDROSTATIC PRESSURE ON THE 

SURVIVAL OF POLIOVIRUS SEEDED IN GROUNDWATER 

AND SEAWATER 250 

Introduction 250 

Materials and Methods 251 

Virus and Viral Assays 251 

Water Samples 251 

Poliovirus Exposure to Hydrostatic Pressures 252 ' 

Results and Discussion 254 

VIII CONCLUSIONS - 258 

APPENDIX: COMPOSITION OF MEDIA AND SOLUTIONS USED ' 

IN ENTEROVIRUS ASSAYS 260 



BIBLIOGRAPHY 



265 



BIOGRAPHICAL SKETCH ___ 285 



VI 



Abstract of Dissertation Presented to the Graduate Council 
of the University of Florida in Partial Fulfillment of the 
Requirements for the Degree of Doctor of Philosophy 



FATE OF VIRUSES FOLLOWING SEWAGE SLUDGE 
APPLICATION TO SOILS 



By 

Oscar Carlos Pancorbo 
August, 1982 



Chairman: Gabriel Bitton 

Major Department: Environmental Engineering Sciences 



In recent years, land disposal of sewage sludge has been viewed 
as a viable alternative to other disposal practices. However, there 
is growing concern over the contamination of groundwater and surface 
waters with microbial pathogens, particularly viruses, present in 
digested sludge. The major objective of this study was to assess the 
potential health risk, from viral pathogens, of sludge application to 
soils. 

Poliovirus type 1 (LSc) was found to be largely associated with 
digested, conditioned-dewatered, chemical and lime-stabilized, chemical- 
sludge solids. Sludge type was found to affect, however, the degree 
of association between seeded poliovirus and sludge solids. For 
example, the degree of association between poliovirus and sludge solids 



vn 



>?f»ig»-.% ' 



was significantly greater for aerobically digested sludges (95%) 
than for activated sludge mixed liquors or anaerobically digested 
sludges (72% and 60%, respectively). The effectiveness of the glycine 
method in the recovery of solids-associated viruses was also found to 
be affected by sludge type. Significantly lower mean poliovirus recovery 
was found for aerobically digested sludges (15%) than for mixed liquors 
or anaerobically digested sludges (72% and 60%, respectively). 

Poliovirus transport studies involving soil cores treated with 
virus-seeded sludge were conducted under controlled laboratory and 
saturated flow conditions. A Red Bay sandy loam displayed a substantially 
greater retention capacity for poliovirus in anaerobically digested sludge 
than a sandy soil (i.e., Eustis find sand). The Red Bay sandy loam was 
shown to completely retain poliovirus following the application of 
conditioned-dewatered, chemical and lime-stabilized, chemical sludge. 

Undisturbed soil cores of Eustis fine sand were treated with several 
inches of virus-seeded (poliovirus and echovirus type 1-Farouk) sludge 
during a two-year period. The soil cores were exposed to natural conditions 
and soil temperature, soil moisture and rainfall were monitored. Both 
viruses were found to be rapidly inactivated in the sludge during the 
drying process on top of the soil cores. Monitoring of the/top inch of 
soil revealed that both viruses were inactivated with time and were 
undetectable after 35 days. Soil leachates collected after natural 
rainfall (unsaturated flow conditions) were negative for both viruses. 

Indigenous enterovirus were not detected in topsoil and groundwater 
samples from two sludge disposal sites in Florida. 



vm 



CHAPTER I 
INTRODUCTION 



In the United States, the Water Pollution Control Act of 1972 
(PL 92-500), as recently amended, requires acceptable methods for the 
utilization and disposal of wastewater effluents and sludges 
(Willems 1976). It now appears that land disposal of wastewater 
effluents and sludges is a viable and attractive alternative to other 
disposal practices. Land spreading of wastewater effluents and sludges 
has many advantages, including the addition of plant nutrients, water 
conservation, improvement of soil physical properties, and increased 
soil organic matter. However, concern has been raised over the con- 
tamination of groundwater and surface waters with nitrates, heavy 
metals, and microbial pathogens, particularly viruses (Bitton 1975; 
Bitton 1980b; Burge and Marsh 1978; Gerba et al. 1975). 

Viruses are generally associated with wastewater solids (Oliver 
1976; Lund 1971) and a significant fraction of these viruses is 
transferred to sludge as a result of wastewater treatment processes. 
Sludge treatment processes, such as anaerobic digestion, do not 
completely inactivate or remove viruses (Bertucci et al. 1977). 
Therefore, the application of anaerobically digested sludge 
onto land can lead to groundwater contamination as a result of virus 
transport through the soil matrix. The movement of sludge-associated 
viruses is probably limited due to the immobilization of sludge solids 

. 1 



in the top portion of the soil profile (Oliver 1976). However, there 
are "free" viruses which have not become associated with the sludge 
solids or which dissociated from these solids as a result of changes 
in the physico-chemical properties within the soil matrix, and which 
may move through the soil to contaminate groundwaters. The movement 
of these individual particles through the soil has been reviewed by 
Bitton (1975) and Gerba et aj^. (1975), and is dependent on the type of 
soil, flow rate, degree of ^saturation of pores, pH, conductivity, and 
the presence of soluble organic materials. 

A multidisciplinary project designed to study the effect of 
sludge application on crops, land, animals, and groundwater was under- 
taken by researchers at the University of Florida, and was funded by 
the U.S. Environmental Protection Agency (Edds et al. 1980). A virus 
study was included since virtually little is known on the survival and 
movement of sludge-bound viruses in soils. The major objective of this 
study was to assess the potential health risk, from viral pathogens, of 
sludge application to soils. This objective was achieved by studying 
the following: 

1. Effect of sludge type on poliovirus association 
with and recovery from sludge solids (Chapter III) 

2. Poliovirus transport studies involving soil cores 
treated with virus-seeded sludge under laboratory 
conditions (Chapter IV) 

3. Retention and inactivation of enteroviruses in soil 
cores treated with virus-seeded sludge and exposed 
to the North-Central Florida environment (Chapter V) 



4, Monitoring of indigenous enteroviruses at two 
sludge disposal sites in Florida (Chapter VI) 

5. Effect of hydrostatic pressure on the survival of 
poliovirus seeded in groundwater and seawater (Chap- 
ter VII~this research was conducted in order to 
determine virus survival in the groundwater 
environment) . 

This research has allowed the determination of the persistence and 
possible movement of pathogenic viruses in soils treated with wastewater 
sludge. The information gained from this study is of value in the 
ultimate assessment of the potential risk of viral infection to humans 
associated with land disposal of sludges. 



CHAPTER II 
LITERATURE REVIEW 

Viral Pathogens Found in Raw Wastewater 
The pathogens found in raw wastewater fall into one of the 
following four groups: bacteria, protozoa, helminthic parasites, and 
viruses. Several reviews have appeared in the literature that address 
all the pathogens found in sewage (Burge and Marsh 1978; Foster and 
Engelbrecht 1973; Elliott and Ellis 1977). Herein, the emphasis will be 
on the viral pathogens present in raw wastewater. 

Over 100 types of viruses are found in raw wastewater (Bitton 
1980b; Burge and Marsh 1978; Foster and Engelbrecht 1973; Elliott and 
Ellis 1977). The most important virus groups are the enteroviruses 
(i.e., polioviruses, coxsackieviruses and echoviruses) , reoviruses, 
adenoviruses, infectious hepatitis agent (viral hepatitis type A--Hall 
1977), and viral gastroenteritis agents [variously designated as duo- 
viruses, rotaviruses, reovirus-like agents or Norwalk agent (parvo- 
virus)— Chanock 1976]. These organisms cause such diseases as polio- 
myelitis, aseptic meningitis, myocarditis, enteritis, jaundice, 
infectious hepatitis, and gastroenteritis (Burge and Marsh 1978; Foster 
and Engelbrecht 1973). 

Indigenous virus concentrations ranging from 500 to 80,000 
plaque-forming units (PFU)/il were measured by Buras (1974) in raw sewage 
from Haifa, Israel. Dugan et al. (1975) found between 27 and 19,000 



PFU/£ of virus in raw sewage from the Mililani (Oahu, Hawaii) sewage 
treatment plant. Mack et al. (1962) found a maximum of 62,800 PFU/il 
of virus in raw sewage. In Austin, Texas, raw sewage, Moore et al . 
(1977) reported virus concentrations between 140 and 1,490 PFU/A. 
Wellings et al_. (1974, 1975) found virus concentrations ranging from 
54 to > 161 PFU/£ in raw sewage from two locations in Florida. Clearly, 
a wide range of virus concentrations and types is found in raw waste- 
water. The virus concentration detected in raw wastewater depends on 
the geographical location, season of the year (Lund et a]_. 1969) and 
virus recovery method used (Buras 1974; Foster and Engelbrecht 1973). 

Removal of Viruses by Wastewater Treatment Processes 
Wastewater treatment processes (Fair et a^. 1968; Zoltek and 
Melear 1978) vary in their ability to remove pathogenic viruses. 
Several authors have reviewed the literature on virus removal by waste- 
water treatment processes (Berg 1973a; Elliott and Ellis 1977; Foster 
and Engelbrecht 1973; Grabow 1968; Kollins 1966; Malina 1976; Sproul 
1976). In this section, the emphasis will be placed on the two treat- 
ment processes that generate the most sludge. These processes are 
primary sedimentation, with or without chemical addition, and acti- 
vated sludge (i.e., secondary treatment). 

Primary Sedimentation 

Primary sedimentation is the most common and sometimes the only 
treatment prior to final disposal of wastewater (Kollins 1966; 
Grabow 1968). The detention time of wastewater in this treatment 



process is usually only some hours (Fair et al^. 1968). The capacity 
of primary sedimentation to remove viruses is at best minimal (Foster 
and Engelbrecht 1973; Kollins 1966; Grabow 1968; Sproul 1976). Clarke 
et a^. (1961) found only 3% removal of seeded poliovirus type 1 
(Mahoney) from raw sewage during a three-hour settling period. Chemical 
flocculation using alum, ferric chloride or lime followed by sedimenta- 
tion (i.e., intermediate or chemical treatment), however, has been shown 
to be very effective in the removal of viruses from raw sewage (Berg 
1973a; Lund 1976; Malina 1976; Grabow 1968; Sproul 1976). The removal 
of viruses by chemical flocculation has been tested in the laboratory 
using suspending media of varying composition (see review by Berg 1973a). 

Several laboratory studies, using suspending media other than 
raw sewage, have shown that seeded viruses are effectively removed 
during flocculation. Change et al. (1958) reported 86.3 to 98.7% removal 
of seeded coxsackievirus A2 after flocculation at pH 6.2 in distilled 
water-Si02-NaHC03 buffer with 40 to 100 ppm of alum [as Al2(S04)3], 
respectively. The removal of this virus by alum flocculation conformed 
to the Freundlich isotherm and, therefore, these investigators concluded 
that the removal mechanism was adsorption. Approximately 60% of the 
virus associated with the aluminum floes was. recovered following 
elution with 0.1 M NaHC03, ^* ^ ^^"^^ P" ^'^ ^^^^ ^^^"9 et al. 1958). 
In a similar study involving phosphate precipitation from water, Brunner 
and Sproul (1970) found 89 to >98% removal of seeded poliovirus type 1 
(Sabin) from distilled water-phosphate medium following alum [68 mg/5, 
as Al2(S0^)2] flocculation. The removal measured was attributed to the 



■ W-: 



adsorption of the virus to the aluminum phosphate floes and, therefore, 
the ranoval efficiency increased with a corresponding increase in the 
quantity of phosphate precipitated. These investigators also found 
that the removal efficiency of poliovirus was influenced by pH with 
maximum removal (i.e.., >96%) observed at pH 6.4, and a reduction in 
removal at pH 5.1 and 7.3. Moreover, only 40% of the polioviruses 
associated with the aluminum phosphate floes were recovered when the 
floes were dissolved in 0.1 N NaOH, final pH 8.3 to 9.3 (see Brunner and 
Sproul 1970). These investigators attributed their lack of recovery 
of the adsorbed viruses to viral inactivation during the precipitation 
process. However, it is likely that the method used to dissolve the 
floes was inefficient in the recovery of the adsorbed viruses. Brunner 
and Sproul (1970) also studied the removal of poliovirus (93 to >97%) 
from filtered wastewater effluent during phosphate precipitation with 
alum. In this medium, virus removal increased with decreasing pH from 
6.9 to 5.0. This was attributed to an increase in suspended solids 
removal with decreasing pH. Bacteriophages have also been effectively 
removed (93 to >99%) from water following floeeulation with alum 
(Brunner and Sproul 1970; Chang et al. 1958; Chaudhuri and Engelbreeht 
1972; York and Drewry 1974). 

In addition to alum, ferric chloride has also been tested as a 
coagulant in the removal of viruses from water. Chang et al_. (1958) 
reported 96.6 and 98.1% removal of seeded coxsackievirus A2 after 
floeeulation at pH 6.2 in distilled water-Si02-NaHC03 buffer with 20 and 
40 ppm of ferric chloride (as FeCl^), respectively. Similarly, Sobsey 



et al_. (1977) studied the flocculation of poliovirus type 1 (LSc) by 
ferric chloride. These investigators added the virus to a membrane 
concentrate (i.e., 0.5 M glycine buffer) of turbid estuary water and 
proceeded to concentrate the virus by flocculation. At a concentration 
of 0.001 M FeClj. >99% of the seeded viruses were removed from the 
supernatant at pH values from 3.5 to 7.5. However, maximum precipita- 
tion was observed at pH 3.5. Bacteriophages have also been removed 
efficiently from water (>99% removals) by flocculation with ferric 
chloride (Change etal^. 1958; York and Drewry 1974). In fact, bacterio- 
phage removals consistently exceeded those of enteroviruses (Chang et al. 
1958). Chang et aX. (1958) found ferric chloride to be more efficient, 
on a molar basis, than alum in the removal of coxsackievirus A2 from 
water by flocculation. Furthermore, these researchers noted that the 
floes formed with ferric chloride were more compact and settled more 
rapidly than those formed with alum. Chang et al_. (1958) were unsuc- 
cessful in recovering the coxsackieviruses associated with the iron 
floes by eluting with 0.1 M NaHCO^, final pH 8.5. It is likely that 
this solution was inadequate as an eluent for the recovery of the 
adsorbed viruses, although viral inactivation during the flocculation 
process cannot be ruled out. In contrast, 74% of the polioviruses asso- 
ciated with the iron floes were recovered by Sobsey et al^. (1977) follow- 
ing elution with fetal calf serum (PCS) at pH 8.0. 

Calcium hydroxide (i.e., lime) is another chemical frequently 
used in flocculation tests for the removal of viruses from water. 
Sproul (1972) has reviewed the literature on virus removal by water- 
softening precipitation processes involving lime as the coagulant. 



'^J^ET^ 



Removals in excess of 99% have been reported for poliovirus type 1 
during excess lime-soda ash softening at pH 10.8 to 11.2 (Wentworth 
et al_. 1968). Enteroviruses are actually inactivated under the high 
pH conditions achieved rather than merely removed by the flocculation 
process (Sproul 1972). The inactivation of enteroviruses under alka- 
line pH is believed to be caused by the denaturation of the protein 
coat and the subsequent disruption of the structural integrity of the 
virus (Sproul 1972). Lime flocculation followed by sedimentation or/and 
sand filtration is frequently used as a tertiary (advanced) treatment 
of secondary wastewater effluents (Malina 1976). This treatment 
effectively removes suspended solids and phosphates from secondary 
effluents (Berg etal. 1968). Several studies (field and laboratory) 
have been undertaken to determine the removal efficiency of viruses from 
secondary effluents by lime flocculation. Brunner and Sproul (1970) 
performed laboratory precipitation tests involving the addition of 
lime [as Ca(0H)2] to filtered wastewater effluent until the pH was 
raised to between 9.5 and 10.9. Poliovirus type 1 (Sabin) removals 
ranging from 88 to 94% were achieved. Generally, virus removal 
increased with a corresponding increase in pH and in the quantity of 
phosphate precipitated. In a similar laboratory study. Berg et al_. 
(1968) showed that 70 to >99% of poliovirus type 1 (LSc) seeded in 
secondary wastewater effluent was removed by lime flocculation [with 
300 mg/Z (pH 10.2) to 500 mg/£ (pH 11.0) of Ca(0H)2, respectively] 
followed by sedimentation. These investigators also observed that 
virus removal increased as the pH achieved by the addition of lime 
increased. Additional experimentation confirmed that poliovirus was 



10 



inactivated by the high pH produced and that the viral inactivation 
rate increased as the pH was raised from 10.1 to 11.1 (see Berg et al_. 
1968). Therefore, these researchers concluded that the removal of 
poliovirus by this treatment process resulted from a combination of 
viral inactivation and physical separation of the virus by the floccu- 
lation process. The effectiveness of lime flocculation in the removal 
of viruses from secondary effluents has also been shown in the field. 
At a wastewater reclamation plant, Grabow et al_. (1978) showed that >99.9/ 
of indigenous enteric viruses in activated sludge effluent were removed 
by lime flocculation at pH 9.6 to 11.2 followed by sedimentation. 
Coliphages, enterococci and coliform bacteria were also effectively 
removed by the lime flocculation process (see Grabow et al_. 1978). 

Chemical flocculation using alum, ferric chloride or lime is 
frequently combined with sedimentation in the primary treatment of raw 
sewage (U.S. Environmental Protection Agency 1973). Among the advantages 
of this treatment process are low capital costs, minimal space require- 
ments and high reliability (Weber etal_. 1970). In laboratory-scale 
experiments, Shuckrow et al_. (1971) showed that the flocculation of 
raw sewage with alum was highly efficient in removing suspended solids, 
total organic carbon (TOC) and chemical oxygen demand (COD). Similar 
results were obtained by Weber et al_. (1970) when lime or ferric chloride 
were used as coagulants. Substantial removals of phosphates, nitrates, 
and organic color are also achieved by this treatment process (Sproul 
1976; Weber et al_. 1970). As reviewed in the previous paragraphs, 
numerous studies have shown that viruses are effectively removed from 



11 



water and wastewater effluents by flocculation with alum, ferric 
chloride or lime. Unfortunately, only a few studies have been con- 
ducted to determine the effectiveness of chemical flocculation in 
removing viruses from raw sewage (see reviews by Berg 1973a; Lund 1976; 
Malina 1976; Sproul 1976). In laboratory-scale pilot plants, Lund and 
R0nne (1973) studied the fate of indigenous enteric viruses in raw 
sewage following flocculation with alum [75 to 175 mg/£ as Al2(S0,)o], 
ferric chloride (25 to 35 mg/£ as FeCl^) or lime (added lime until pH 
10.5 was maintained). These investigators noted appreciable removals 
of viruses in all flocculation experiments. The viruses were found 
concentrated in the chemical sludges produced. Furthermore, no viral 
inactivation was observed in the chemical sludges. In another labora- 
tory study, Sattar et al_. (1976) showed that 99.995% of poliovirus 
type 1 (Sabin) seeded in raw sewage was inactivated during flocculation 
with lime at pH 11.5, No viruses were recovered in the supernatants 
following flocculation and an average of only 0.005% of the total 
viral input was recovered in the lime sludges by eluting with 10% PCS 
in saline, pH 7.2. During storage at 28°C, further viral inactivation 
was noted in the lime sludges and no viruses could be detected in the 
sludge samples after 12 hours. The inactivation rate of seeded polio- 
virus type 1 (Sabin) during lime flocculation of raw sewage was 
reduced as the pH of the solution was decreased. Sattar and Ramia 
(1978) found that 92.4 and 97.2% of the total inputs of poliovirus 
were inactivated (i.e., recovered only 7.6 and 2.8% in the supernatants 
and sludges combined) during lime flocculation at pH 9.5 and 10.5, 



12 



respectively. In these studies (Sattar et a^, 1976; Sattar and Ramia 
1978), it was pointed out that the method used (i.e., elution with 
10% FCS in saline, pH 7.2) to recover viruses from the lime sludges 
may not have been completely effective. In spite of this possible 
limitation, the authors concluded that substantial viral inactivation 
can be achieved during the flocculation of raw sewage with lime at high 
pH. 

The literature presented herein indicates that chemical floccu- 
lation, under optimal laboratory conditions, can remove large quanti- 
ties of viruses from raw sewage and from other water samples. Virus 
removal efficiency depends on the coagulant, coagulant dose, water 
type, pH, virus type and flocculation procedure employed. Since it 
is difficult to ensure optimum floe formation routinely in practice, 
Grabow (1968) hypothesized that, under field conditions, significant 
virus removals by flocculation are not likely. As proposed by Berg 
(1973b), more research is needed before a conclusion can be reached as 
to the virus removal capacity of chemical flocculation in actual prac- 
tice. 

The effluents from the primary sedimentation units generally 
still contain demonstrable quantities of enteric viruses (Sattar and 
Ramia 1978). These primary effluents usually undergo further treatment 
by, for example, the activated sludge process. Large quantities of 
sludge (i.e., 2,400 to 5,000 gallons of sludge per million gallons 
of wastewater treated) are produced during the primary sedimentation 
process (U.S. Environmental Protection Agency 1974). When chemical 



13 



flocculation is combined with primary sedimentation, even larger quan- 
tities of sludge (i.e., 5000 to 38,000 gallons of sludge per million 
gallons of wastewater treated) are produced (U.S. Environmental Protec- 
tion Agency 1974). These raw primary sludges (in particular, the 
chemical sludges) contain significant amounts of enteric viruses (Berg 
and Berman 1980; Lund 1976; Lund and R^nne 1973; Nielsen and Lydholm 
1980; Sattar and Ramia 1978; Sproul 1976). In fact, the indigenous 
enteric viruses are concentrated in the chemical sludges (Lund and R0nne 
1973), Sattar and Ramia (1978) readily detected indigenous viruses in 
all the primary lime sludge samples they tested. The sludge samples 
were obtained from a wastewater treatment plant in Canada which employed 
lime flocculation of raw sewage at a pH of approximately 10. Clearly, 
even under such virucidal conditions of alkaline pH, viruses were 
recovered from the sludges produced. Thus, all primary sludges should 
be considered to represent potential health hazards, and should be 
handled with care during further treatment and final disposal (Brunner 
and Sproul 1970; Lund 1976; Sattar and Ramia 1978). 

Activated Sludge 

The activated sludge process (i.e., secondary or biological treat- 
ment) is perhaps the most effective wastewater treatment process in the 
removal of viruses. Several authors have reviewed the literature on virus 
removal by this treatment process (Berg 1973a; Foster and Engelbrecht 1973; 
Grabow 1968; Kollins 1966; Malina 1976; Sproul 1976). In laboratory- 
scale pilot plants, 90% or more of the seeded enteroviruses were removed 
by the activated sludge process (Clarke et al. 1961; Malina et al_. 1975). 



14 



In these studies, the seeded viruses were transferred to the settled 
sludges (Clarke etal. 1961; Malina etal. 1975). Clarke etal. (1961) 
recovered only a small fraction of the viruses (i.e., poliovirus type 
1— Mahoney or coxsackievirus A9) theoretically associated with the 
settled sludge. These researchers concluded that the viruses were 
inactivated when adsorbed to sludge particles. However, their failure 
to adequately recover the adsorbed viruses can partly be attributed 
to the poor recovery method used (i.e., used buffer solutions and 
versene as eluents). In a laboratory-scale activated sludge unit, 
Malina et a]_. (1975) effectively recovered poliovirus type 1 (Mahoney) 
from the settled sludge by using a better eluent, distilled deionized ■ 
water. Moreover, poliovirus associated with the sludge particles was 
observed to be inactivated over time. These investigators measured the 
viral inactivation rate in the settled sludge and found that the rate 
conformed to the following equation: 

Ct = ^-e-'^2t (2-1) 

where C^ = virus associated with sludge initially (PFU/mg of 

dry sludge solids) 
C^ = virus associated with sludge at time t (PFU/mg of 

dry sludge solids) 
t = time (min) 
kg = rate constant (min" ) 

The rate constant (i.e., k^) varied with mixed liquor suspended solids 
(MLSS) concentration. For example, rate constants of 3.17 x 10'^ and 



15 



-3 -1 
2.5 X 10 min were determined for MLSS concentrations of 1,590 and 

3,140 mg/£, respectively (see Malina et aj^. 1975). 

As far as indigenous viruses are concerned, much less work has 
been done on their removal by the activated sludge process. Moore et 
a^. (1977, 1978) and Farrah et al_. (1981b) readily recovered indigenous 
• enteroviruses from the mixed liquor suspended solids of activated sludge 
plants in Texas, Illinois, Montana and Oregon, and Florida, respectively. 
Since in these three studies most of the viruses detected were directly 
associated with the solids, it can be hypothesized that a large frac- 
tion of these viruses would be removed during subsequent secondary 
sedimentation of the sludge solids. The effectiveness of the activated 
sludge process in removing indigenous viruses from wastewater was 
confirmed by Lund et al_. (1969) and Moore et al. (1977) in field studies 
in Denmark and the United States (Austin, Texas), respectively. 

In. spite of the substantial virus removal capacity of the 
activated sludge process, effluents from this treatment method routinely 
contain demonstrable indigenous viruses (Buras 1974; Dugan et al^. 
1975; England etal_. 1965; Gilbert etal. 1976a, 1976b; Merrell and 
Ward 1968; Moore et al. 1977; Vaughn et al. 1978; Wellings et al. 
1974, 1976a, 1978). Further treatment by chlorination (Dugan et al. 
1975; England et al. 1965; Merrell and Ward 1968; Vaughn et al. 1978; 
Wellings et al. 1974, 1978), or by tertiary processes such as oxidation 
pond, denitrification followed by sand filtration, or alum floccula- 
tion (England etal. 1965; Merrell and Ward 1968; Vaughn et al. 1978; 
Wellings et al. 1978) often does not eliminate all indigenous viruses 
from activated sludge effluents. In addition to viruses, secondary 



16 



effluents also contain bacterial pathogens (Foster and Engelbrecht 
1973), parasites (Hays 1977) and a variety of hazardous chemicals 
(e.g., nitrates, phosphates, chromium, cadmium, mercury, zinc, copper, 
polychlorinated biphenyls and phthalates; see Lee 1976). Therefore, 
secondary effluents should be regarded as potential health hazards in 
their final disposal . 

During the activated sludge process, large quantities of sludge 
(i.e., 14,000 to 19,000 gallons of sludge per million gallons of 
wastewater treated) are also produced (U.S. Environmental Protection 
Agency 1974). The volume of waste activated sludge (i.e., secondary 
sludge) produced is usually much greater than the volume of sludge gener- 
ated during primary sedimentation, due to the greater moisture content 
of the former (U.S. Environmental Protection Agency 1974). In fact, 
when a wastewater treatment plant is upgraded to activated sludge 
treatment, the capacity for excess sludge handling (i.e., sludge 
treatment and disposal) must be significantly increased (U.S. Environ- 
mental Protection Agency 1974). Indigenous enteric viruses are 
routinely found in raw secondary sludges (Berg and Berman 1980; Lund 
1976; Lund and R0nne 1973; Nielsen and Lydholm 1980). Therefore, all 
secondary sludges should be considered to represent potential health 
hazards, and should be handled with care during further treatment and 
final disposal. 

Removal of Viruses by Sludge Treatment Processes 
Viruses in Raw Sludges 

As reviewed above, raw sludges produced during the primary and 
secondary treatment of wastewater contain substantial quantities of 



^ V'-^ITV^^^- 



V 



enteric viruses. The indigenous virus titer of raw sludge has been 
measured by several investigators and expressed in different units. 
In raw primary sludge, indigenous enteric virus concentrations of 2.4 
to 15 PFU/ml, ^.9 X 10^ PFU/ml , 6.9 to 215 PFU/g dry wt. of total 
suspended solids (TSS), 7.9 x 10^ to 4.3 x 10^ PFU/g dry wt. TSS and 
10 to 1,000 50% tissue culture infective dose (TCIDcQ)/ml were measured 
by Oliver (1975), Nath and Johnson (1980), Turk et al. (1980), Moore et 
al. (1978) and Lund (1976), respectively. In mixtures of primary 
(1/3) and secondary (2/3) raw sludge from the City of Los Angeles 
Hyperion treatment plant. Berg and Berman (1980) found indigenous 
enteric virus concentrations ranging from 3.8 to 116 PFU/ml. Nielsen 
and Lydholm (1980) detected 0.1 to 9.0 TCID^Q/mg TSS of indigenous 
enteric viruses in raw sludge (primary and secondary) from three 
Danish wastewater treatment plants. Secondary sludges have been shown 
to contain 10- to 100-fold less virus than primary sludges (Lund 1976; 
Lund and R0nne 1973). Clearly, a wide range of virus concentrations is 
found in raw sludges. The indigenous virus concentration detected in 
raw sludge depends on the sludge type, geographical location, season 
of the year (Berg and Berman 1980) and virus recovery method used (Nath 
and Johnson 1980). Moreover, a large fraction of the indigenous 
viruses in raw sludges have been shown to be associated with the sludge 
solids (Oliver 1975; Lund 1976; Lund and R0nne 1973; Nath and Johnson 
1980). Indigenous enteric viruses are strongly associated with fecal 
solids in raw wastewater (Bitton 1980a; Oliver 1975; Oliver 1976; 
Wellings et al_. 1976a) and tend to remain associated with solids during 



18 



wastewater treatment processes (Cliver 1976). Furthermore, 
indigenous viruses are believed to be mostly embedded within the 
sludge solids rather than merely surface adsorbed (Wei lings et al. 
1976a). This association between indigenous viruses and sludge solids 
has significant implications on viral survival during subsequent 
sludge treatment. 

Sludge Treatment Processes 

A variety of sludge treatment processes is routinely used in 
treatment plants and they are shown in Figure 2-1. From Figure 2-1, it 
can also be seen that several often overlapping functions are achieved 
by each sludge treatment process. The order of the sludge treatment 
processes shown in Figure 2-1 is as most often used in treatment 
plants (Fair et al. 1968; Malina 1976; U.S. Environmental Protection 
Agency 1974). However, it should be pointed out that not all sludge 
treatment processes shown in Figure 2-1 are employed at e\/ery treatment 
plant. Moreover, the order of the sludge treatment processes may be 
varied from that shown in Figure 2-1 depending on the existing condi- 
tions. Characteristics of the sludge treatment processes have been 
reviewed by several authors (Dick 1978; Fair et al. 1968; Malina 1976; 
U.S. Environmental Protection Agency 1974, 1978a, 1978b; Yates 1977). 
Some work has been done on the removal of viruses (i.e., by physical 
separation and/or inactivation of viral particles) from raw sludges 
(i.e., primary, chemical, and/or secondary sludges) by sludge treatment 
processes and is reviewed below. 



FIGURE 2-1. Outline of sludge treatment processes and their functions 

Adapted from Fair et al- (1968), Malina (1976) and 
United States Environmental Protection Agency (1974, 
1978a, 1978b). 



20 



TREATMENT PROCESSES 
Raw Sludge ^ 



FUNCTIONS 



THICKENING 




1. Water removal (i.e., in- 

crease in solids content) 

2. Volume reduction 

3. Blending (e.g., primary 

with secondary sludge) 


1. Gravity settling 

2. Flotation 

3. Centrifugation 




_... ^ 


f 






STABILIZATION 




1. Pathogen destruction 

2. Odor control 

3. Putrescibility control 

4. Volatile sludge solids 

reduction 

5. Volume reduction 


1. Anaerobic digestion 

2. Aerobic diaestion 


3. Composting 

4. Lime or en 

5. Heat treat 


lorine treatment 
ment 





I 



CONDITIONING 



Chemical (ferric chloride, 
ferrous sulfate, lime, alum 
or organic polymers) treat- 
ment 

Heat treatment 



Improves subsequent 
dewatering 
Stabilization 



DEWATERING 



1. Rotary vacuum filter 

2. Centrifugation 

3. Drying beds 

4. Drying lagoons 



I 



1 . Water removal 

2. Volume reduction 

3. Reduces fuel requirements 
for incinerating/drying 



HEAT DRYING 




1 . Water removal 

2. Volume reduction 

3. Sterilization 


1 . Flash dryer 

2. Tray dryer 

3. Spray dryer 

4. Multiple hearth 




> 


' 






REDUC 


noN 




1. Volatile sludge solids 

destruction 

2. Water removal 

3. Volume reduction 

4. Sterilization 


1. Incineration 

2. Wet air oxidation 

3. Pyrolysis 




. i 


*" 






FINAL DISPOSAL 




1. Disposal (ocean and land- 

fill) 

2. Utilization (i.e., land 

reclamation or use on 
cropland) 


1 . ' Ocean disposal 

2. Sanitary lanrlfili 


3. Land appli 


cation 





21 



Stabilization-digestion . Raw sludges are frequently stabilized 
using anaerobic (Fair et al- 1968; U.S. Environmental Protection Agency 
1974, 1978a) or aerobic (U.S. Environmental Protection Agency 1974, 
1978a; Yates 1977) digestion. These digestion processes reduce the 
odor, putrescibility potential and pathogen content of raw sludges 
(U.S. Environmental Protection Agency 1974, 1978b) and thereby achieve 
sludge stabilization (see Figure 2-1). 

There are several different design-types of anaerobic sludge 
digestion and they employ either no heating, heating at mesophilic 
(30-35°C) temperatures, or heating at thermophilic (approximately 50°C) 
temperatures (U.S. Environmental Protection Agency 1974). In general, 
the rate of sludge stabilization increases as the anaerobic digestion 
temperature is increased. Consequently, the sludge digestion time 
is usually reduced as the anaerobic digestion temperature is increased 
(U.S. Environmental Protection Agency 1974). 

Several authors have reviewed the literature on viral inactiva- 
tion during anaerobic digestion of raw sludges (Berg 1973a; Bitton 1978; 
Cliver 1976; Foster and Engelbrecht 1973; Moore et al. 1977, 1978). In 
laboratory and field studies, enteroviruses have been shown to be in- 
activated during anaerobic digestion (Bertucci et al. 1977; Berg and 
Berman 1980; Cliver 1975; Eisenhardt et al. 1977; Moore et al. 1977, 
1978; Nielsen and Lydholm 1980; Palfi 1972; Sattar and Westwood 1979). 
During anaerobic digestion (35°C) of sludge in laboratory-scale units, 
Eisenhardt etal. (1977) measured the inactivation rate of seeded 



22 



coxsackievirus B3 at 2 log^Q units per 24 hours. In similar laboratory 
anaerobic digesters, Bertucci et al- (1977) observed the inactivation 
rates of enteroviruses [i.e., poliovirus 1 (Sabin), coxsackievirus A9 
(Griggs), coxsackievirus 34 (JVB) and echovirus 11 (Gregory)] seeded in 
sludge to follow a first-order reaction pattern and to significantly 
differ for the four viruses tested. 

Ward and his collaborators have done a great deal of work on 
the inactivation of enteric viruses seeded in raw and anaerobically 
digested sludges. Poliovirus type 1 (CHAT) and poliovirus type 
1 (Mahoney) seeded in anaerobically digested sludge (6% sludge solids, 
pH 8.0) became largely associated (67 and 65% of the total virus 
added, respectively) with the sludge solids and both were inacti- 
vated at rates of approximately 1 log^Q units/5 days at 4°C, >2 log,Q 
units/3 days at 20°C, and >1 log^Q units/day at 28°C (Ward and 
Ashley 1976). Anaerobically digested sludge displayed no detectable 
virucidal activity against enteroviruses when adjusted to pH values 
between 4.5 and 7.5 (Ward and Ashley 1977a). Similarly, there was no 
appreciable inactivation of seeded poliovirus type 1 in 5 days at 20°C 
in raw sludge maintained at its naturally low pH of 6.0 (Ward and 
Ashley 1976). However, the virucidal activity against poliovirus in 
raw sludge significantly increased as the pH of the sludge was raised 
above 7.5 (Ward and Ashley 1977a). Thus, Ward and Ashley (1977a) 
demonstrated that the uncharged form of ammonia, which exists mostly 
at pH values above 8, was the causative agent in the irreversible 
inactivation of enteroviruses in anaerobically digested sludge and in 



23 



raw sludge adjusted to pH values above 7.5. Moreover, this agent was 
shown to be present mainly in the sludge supernatant (i.e., produced 
by centrifuging anaerobically digested sludge at 18,000 x g for 20 
minutes) rather than in the sludge solids (Renters et al_. 1979; Ward 
and Ashley 1976). Microbial activity in anaerobically digested sludge 
supernatant was found not to affect the inactivation rates of seeded 
poliovirus 1 (Sabin), echovirus 6 and coxsackievirus B4 (Fenters et al. 
1979). The mechanism of inactivation of poliovirus type 1 (CHAT) in 
anaerobically digested sludge was found to be cleavage of the two 
largest viral coat proteins (i.e., breakdown of VP-1 and VP-2) followed 
by nicking of the encapsulated RNA (Ward and Ashley 1976). At the 
thermophilic temperature of 43°C, the inactivation rate of seeded 
poliovirus type 1 (CHAT) was significantly lower in raw and anaerobi- 
cally digested sludge than in phosphate-buffered saline (PBS) (Ward 
et al_. 1976). Ward et al_. (1976) proposed that poliovirus was protected 
from heat inactivation by a component found in the sludge solids. In 
the case of anaerobically digested sludge, however, this protective 
effect was always less than that observed for raw sludge and was 
largely reversed at higher temperatures (i.e., 47 and 51°C) due to the 
presence of the virucidal agent, uncharged ammonia (Ward et al. 1976). 
Thus, at the higher temperatures, the inactivation rates of poliovirus 
in anaerobically digested sludge were similar to those in PBS (Ward 
et aX. 1976). Ward et al. (1976) also demonstrated that enteroviruses 
in anaerobically digested sludge were irreversibly inactivated (RNA 
molecule hydrolyzed) during heating at temperatures of approximately 



24 



50°C. At the low natural pH of the raw sludge, the virucidal agent 
was not present (Ward and Ashley 1976, 1977a). Therefore, only the 
protective effect attributed to the sludge solids was observed for 
this sludge type at all temperatures tested (i.e., 43 to 51°C) (Ward 
et al_. 1976). Ionic detergents were later identified as the components 
in raw and anaerobically digested sludge solids which protected polio- 
virus type 1 (CHAT) and other enteroviruses from heat inactivation 
(Ward and Ashley 1977c, 1978a, 1979). In contrast, these ionic deter- 
gents (cationic more active than anionic) were shown to be responsible 
for reducing the heat required to inactivate reovirus type 3 (Dearing) 
in raw and anaerobically digested sludge (Ward and Ashley 1977c, 1978a). 
Ward and Ashley (1979) demonstrated that two ionic organic detergents, 
sodium dodecyl sulfate and dodecyltrimethyl ammonium chloride in buffer 
solutions, were potent virucidal agents for reovirus, but that their 
virucidal effects were strongly pH dependent. The virucidal activity 
against reovirus displayed by these ionic detergents was greater in 
alkaline than in acid conditions (Ward and Ashley 1977c). Further 
research (Ward and Ashley 1979) revealed that the inactivation pattern 
of reovirus as a function of pH at 45°C in anaerobically digested 
sludge was qualitatively similar to that found in buffer solutions 
containing ionic detergents. 

The literature reviewed in the paragraph above indicates that 
the inactivation of enteric viruses in anaerobically digested sludge is 
a complex phenomenon. It is clear, however, that the anaerobic diges- 
tion process raises the pH of raw sludge and thereby produces in the 



25 



digested sludge the uncharged form of ammonia which is virucidal for 
enteroviruses. The presence of this virucide coupled with thermophilic 
digestion temperatures (i.e., approximately 50°C) results in the 
rapid inactivation of enteroviruses in anaerobically digested sludge. 
Furthermore, the protective effect towards enteroviruses attributed to 
ionic detergents in the sludge solids and observed at low pH in raw 
sludge is largely overcome by the virucidal ammonia in anaerobically 
digested sludge (Ward and Ashley 1977c) particularly at thermophilic 
temperatures. Reoviruses are also inactivated in anaerobically digested 
sludge at thermophilic temperatures and at the naturally high pH values 
attained by the digestion process (Ward and Ashley 1979). 

As presented above, the inactivation of viruses during anaerobic 
digestion has been studied in the laboratory using sludge artificially 
contaminated with virus. The validity of such research has been ques- 
tioned because it is believed that, unlike indigenous viruses, the 
seeded viruses become mostly adsorbed to the surface of sludge solids 
(Moore et aj[. 1977; Nielsen and Lydholm 1980). Due to their strong 
association with fecal solids in raw wastewater (Bitton 1980a; Oliver 
1975, 1976; Wei lings et al. 1976a), indigenous viruses are believed to 
end up, during wastewater treatment, mostly embedded within sludge solids 
rather than merely surface adsorbed (Wellings et al. 1976a). Conse- 
quently, Moore etal_. (1977) proposed that indigenous viruses in sludge 
are less susceptible than seeded viruses to the environmental stresses 
(e.g., chemical and heat inactivation) encountered during sludge treat- 
ment by virtue of the former's more insulated environment. Evidence to 



26 



support this hypothesis has been obtained from studies involving the 
anaerobic digestion of sludge. At the East Pearl treatment plant in 
Boulder, Colorado, Moore et al. (1978) found total reductions of 
indigenous enteroviruses in primary raw sludge of only 2 log,^. units 
(i.e., 99%) during 100 days of anaerobic digestion (40 days at 37°C 
in digester no. 1 followed by 60 days in unheated digester no. 2). 
Significantly higher inactivation rates were measured for seeded entero- 
viruses by Bertucci et al. (1977) and Eisenhardt et al. (1977) during 
anaerobic digestion of sludge. For example, Eisenhardt et al. (1977) 

found the inactivation rate of seeded coxsackievirus B3 to be 2 loq,„ 

^10 

units per 24 hours. The inactivation rates of seeded enteroviruses were 
drastically reduced when the viruses were incorporated into the sludge 
during sludge production rather than simply mixed with the final 
sludge sample. Moore et al. (1977) reported an inactivation rate of 
approximately 2 log^Q units per 15 days for poliovirus naturally 
incorporated (and assumed embedded) into wasted sludge during activated 
sludge treatment in a continuous flow, bench-scale unit and then sub- 
jected to anaerobic digestion at 30°C. In fact, these investigators 
detected poliovirus in the sludge undergoing anaerobic digestion even 
after 30 days. Clearly, enteroviruses embedded within sludge solids 
are afforded some protection from virucidal chemical agents (e.g., 
uncharged ammonia in the liquid fraction of anaerobically digested 
sludge-see Ward and Ashley 1977a) and/or physical stresses (e.g., 
heat) encountered during the anaerobic digestion of sludge, as well as 
during other sludge treatment processes. 



27 



Although the anaerobic digestion process appears capable of 
removing considerable quantities of viruses from sludge, a fraction of 
the viruses initially present will, nevertheless, survive this diges- 
tion process [see reviews by Berg (1973a), Bitton (1978), Oliver 
(1976), Foster and Engelbrecht (1973), and Moore et al_. (1978)]. 
Indigenous viruses have been routinely detected in anaerobically 
digested sludge (Berg and Berman 1980; Cliver 1975; Farrah et al. 1981a; 
Moore et al. 1978; Nielsen and Lydholm 1980; Palfi 1972; Sattar and 
Westwood 1979; Sagik et al. 1980; Turk et al. 1980; Wei lings et al. 
1976a). Moreover, the indigenous virus titer of this sludge type has 
been measured by several investigators and expressed in different 
units. Anaerobically digested sludge sampled at various locations 
throughout the United States displayed indigenous enteric virus con- 
centrations of to 8 PFU/ml, < 0.014 to 4.1 PFU/ml , 7 to 40 PFU/g 
dry wt. TSS, 1.1 to 17 PFU/g dry wt. TSS, 0.2 to 17.0 PFU/g dry wt. TSS 
and 2 to 7 TCID^Q/g dry wt. TSS as measured by Cliver (1975), Berg and 
Berman (1980), Moore et al. (1978), Sagik etal. (1980), Turk et £[. 
(1980) and Farrah et al. (1981a), respectively. Nielsen and Lydholm 
(1980) found to 600 TCID^Q/g dry wt. TSS of indigenous enteric 
viruses in anaerobically digested sludge from three Danish wastewater 
treatment plants. Evidently, the indigenous virus concentration 
detected in anaerobically digested sludge depends on the virus con- 
centration in the raw sludge, and thereby, on the geographical location 
(Sagik et al. 1980) and on the season of the year (Berg and Berman 
1980; Moore etal. 1978). The indigenous virus titer found in 



28 



anaerobically digested sludge also depends on the virus removal 
efficiency of the anaerobic digestion procedure employed. For example. 
Berg and Berman (1980) reported that, at the City of Los Angeles 
Hyperion treatment plant, thermophilic anaerobic digestion (20 days at 
approximately 49°C) was superior to mesophilic anaerobic digestion 
(20 days at approximately 35°C) in the removal of indigenous viruses 
from raw sludge. In the laboratory. Ward et al. (1976) confirmed that 
the inactivation of seeded poliovirus type 1 (CHAT) in anaerobically 
digested sludge was accelerated under thermophilic temperatures. 

Although little is known about the viral-inactivating capacity 
of the aerobic digestion process, several investigators have reported 
that, as in the case of anaerobic digestion, not all indigenous entero- 
viruses are eliminated from sludge during aerobic digestion (Farrah 
etai. 1981a, 1981b; Hurst et al. 1978). Farrah et al. (1981a, 1981b) 
measured indigenous enterovirus titers ranging from 1.7 to 260 TCIDt-«/g 
dry wt. TSS in aerobically digested sludge from three wastewater treat- 
ment plants in Florida. In sludges from two wastewater treatment 
plants in Pensacola, Florida, Farrah et al. (1981a) showed that aerobi- 
cally digested sludge contained larger indigenous viral titers than 
anaerobically digested sludge. 

Clearly, both anaerobically and aerobically digested sludges can 
contain substantial quantities of enteric viruses. Therefore, all 
digested sludges should be considered to represent potential health 
hazards, and should be handled with care during further treatment and 
final disposal (Palfi 1972). 



29 



Stabi 1 1 zation-composti ng . Composting is a biological, aerobic, 
thermophilic (approximately 60°C) process frequently used to stabilize 
(see Figure 2-1) raw sludges (U.S. Environmental Protection Agency 1974, 
1978b). As such, this process reduces the odor, putrescibility poten- 
tial and pathogen content of raw sludges (U.S. Environmental Protection 
Agency 1974, 1978b). Bitton (1980b) described in detail the sludge 
composting process and reviewed the literature on pathogen destruction 
during this sludge treatment procedure. Pathogenic parasites and 
bacteria as well as bacteriophage f2 have been shown to be inactivated 
during sludge composting (Bitton 1980b). Ward and Ashley (1978b) 
demonstrated that seeded poliovirus type 1 (CHAT) was heat-inactivated 
(43°C) at a significantly greater rate in composted sludge than in 
dewatered raw sludge held at the same sludge solids content of 40%. In 
the case of seeded reovirus, the reverse was observed (i.e., greater 
inactivation rate in dewatered raw sludge; see Ward and Ashley 1978b). 
These viral inactivation patterns were attributed to the effects of 
sludge solids-associated, ionic detergents (Ward and Ashley 1978b). 
These detergents were previously shown to influence differently the 
heat inactivation rate of enteroviruses and reoviruses in sludge. 
Whereas enteroviruses in sludge were protected from heat inactivation 
by ionic detergents, reoviruses were inactivated at an accelerated 
rate (Ward and Ashley 1977c, 1978a, 1979). During composting, however, 
the ionic detergents in raw sludge were shown to be substantially 
degraded (Ward and Ashley 1978b). Thus, viral inactivation rates were 
markedly different in composted sludge as compared to raw sludge and 



30 



for enteroviruses versus reoviruses (Ward and Ashley 1978b). Although 
most enteroviruses are rapidly inactivated during sludge composting, 
reoviruses apparently are capable of surviving this sludge treatment 
process (Ward and Ashley 1978b). Clearly, sludge composting does not 
yield a virus-free product, and therefore, all composted sludges should 
be handled with care during further treatment and final disposal. 

Stabilization-lime treatment . Lime treatment at pH 11.0 to 11.5 
is another practice frequently employed to stabilize (see Figure 2-1) 
raw sludges (Farrell et al. 1974; U.S. Environmental Protection Agency 
1974, 1978a). During periods when digesters are out of service or when 
sludge quantities exceed digester design capacity, lime treatment is an 
effective alternate method of sludge stabilization (Farrell et £[. 1974; 
U.S. Environmental Protection Agency 1978a). Due to the large quanti- 
ties of chemical sludges (e.g., alum and iron) usually produced, lime 
treatment is particularly suited for the stabilization of these sludge 
types (Farrell et al_. 1974). At relatively low costs, lime stabilization 
reduces the odor, putrescibility potential and pathogen content of raw 
sludges (Farrell et al. 1974; U.S. Environmental Protection Agency 1974, 
1978a, 1978b). However, the effectiveness of this sludge stabilization 
procedure is apparently dependent upon the pH achieved and maintained. 
Farrell et a]_. (1974) demonstrated adequate stabilization of chemical 
sludges during lime treatment at pH 11.5 for 30 minutes (pH was main- 
tained above 11 for 24 hours). Further research has indicated, however, 
that the pH must be maintained above 12 for 30 minutes (pH remaining 
above 11 for at least 14 days) during liming in order to ensure effec- 
tive sludge stabilization (U.S. Environmental Protection Agency 1974, 



31 



1978a). The lime dosage required to exceed, for example, pH 12 for 
30 minutes has been found to be affected by the sludge type, chemical 
composition of the sludge and percent sludge solids (Farrell et al. 
1974; U.S. Environmental Protection Agency 1978a). In addition to 
achieving stabilization, lime treatment also conditions the sludge (see 
Figure 2-1) such that subsequent sludge dewatering is improved (Farrell 
et al_. 1974; U.S. Environmental Protection Agency 1974). 

Most bacterial pathogens in raw sludge have been shown to be 
destroyed during lime stabilization (Farrell et al. 1974; U.S. Environ- 
mental Protection Agency 1974, 1978a). Fecal streptococci, however, 
remain viable during liming (U.S. Environmental Protection Agency 
1978a). Moreover, regrowth of bacterial organisms can occur if the 
pH of the lime-stabilized sludge is allowed to drop rapidly below 11 
(Farrell et al. 1974; U.S. Environmental Protection Agency 1974). 
Under ideal conditions, lime treatment is superior to anaerobic diges- 
tion in the inactivation of bacterial pathogens in raw sludge (U.S. 
Environmental Protection Agency 1978a). As a result, the bacterial 
pathogen concentrations in lime-stabilized sludges are 10 to 1,000 
times lower than in anaerobically digested sludges (U.S. Environmental 
Protection Agency 1978a). 

As far as enteric viruses are concerned, no research has been 
conducted on their fate during lime stabilization of raw sludge 
(Farrell et al. 1974). However, since seeded poliovirus type 1 has 
been shown to be inactivated during the flocculation of raw sewage with 
lime at pH 11.5 and to be undetectable (i.e., apparently fully 



32 



inactivated) after 12 hours in the lime sludge produced (Sattar et al^. 
1976), it can be hypothesized that substantial quantities of entero- 
viruses are probably inactivated during the lime stabilization of 
raw sludges (i.e., primary, chemical and secondary sludges). This 
hypothesis has yet to be confirmed experimentally. Until new informa- 
tion becomes available, lime-stabilized sludges should be regarded as 
potentially containing pathogenic enteric viruses, and, therefore, should 
be handled with care during further treatment and final disposal. 

Stabilization-heat treatment . Heat treatment is yet another 
process which has been used to stabilize (see Figure 2-1) raw sludges 
(U.S. Environmental Protection Agency 1974, 1978b). Two types of heat 
treatment have been used for sludge stabilization, and they are pasteuriza- 
tion at approximately 70°C and low-pressure (180 to 210 psi) oxidation 
at approximately 200°C (U.S. Environmental Protection Agency 1974). 
Pasteurization at 70°C for 30 to 60 minutes destroys most pathogens 
in raw sludge including enteric viruses (U.S. Environmental Protection 
Agency 1974). Due to the extremely high temperatures (i.e., 200°C) 
employed during low-pressure oxidation, all pathogens, including 
enteric viruses, in raw sludge are undoubtedly destroyed (U.S. Environ- 
mental Protection Agency 1974, 1978b). Under the most ideal conditions, 
however, low-pressure oxidation has displayed a poor capacity to reduce 
the odor and the putrescibility potential of raw sludges (U.S. Environ- 
mental Protection Agency 1978b). As shown in Figure 2-1 and described 
below, heat treatment by the low-pressure oxidation process also 
conditions the sludge such that subsequent sludge dewatering is 



33 



improved (U.S. Environmental Protection Agency 1974, 1978a). It should 
be pointed out that the heat treatment of sludge is an energy-intensive 
process. Consequently, in actual practice, the applicability of this 
treatment procedure is limited due to high energy costs. 

Conditioning-chemical treatment . As shown in Figure 2-1, 
stabilized sludge is frequently conditioned with organic polymers or 
inorganic chemicals (i.e., ferric chloride, ferrous sulfate, lime or 
alum) in order to facilitate water removal by subsequent sludge de- 
watering processes (U.S. Environmental Protection Agency 1974, 1978a). 
These flocculants provide charge neutralization and thereby aggregate 
the sludge particles such that a porous, free-draining cake structure 
is produced (U.S. Environmental Protection Agency 1974, 1978a). Con- 
sequently, chemical conditioning improves sludge dewaterability and 
sludge solids capture during dewatering procedures (U.S. Environmental 
Protection Agency 1974). 

Although never tested, it can be hypothesized that indigenous 
enteric viruses in sludge are probably associated with the sludge- 
particle aggregates produced during the chemical conditioning of sludge. 
Thus, enteric viruses can be expected to be concentrated in the con- 
ditioned-dewatered sludge. Except in the case of lime treatment, no 
significant viral inactivation is likely to result from chemical con- 
ditioning. 

Conditioning of sludge with lime is routinely undertaken in 
conjunction with ferric chloride (U.S. Environmental Protection Agency 
1974). As shown in Figure 2-1 and described above, lime treatment 



34 



also provides stabilization of the sludge (U.S. Environmental Protection 
Agency 1974, 1978a, 1978b). As such, lime conditioning of sludge 
reduces odors and the pathogen, including viral, content of sludge 
(U.S. Environmental Protection Agency 1974, 1978b; also see pages 30 to' 
32 in this chapter). Until more information becomes available, however, 
lime-conditioned sludges should not be regarded as virus free. 

Conditioning-heat treatment . Heat treatment at temperatures of 
300 to 500°F (i.e., 150 to 260°C) and pressures of 150 to 400 psi for 
periods of 15 to 40 minutes is another conditioning process which 
facilitates sludge dewatering (see Figure 2-1) (U.S. Environmental Pro- 
tection Agency 1974, 1978a). Such heat treatment solubilizes and 
hydrolyzes the smaller and more highly hydrated sludge particles which 
are then removed from the bulk sludge sample and end up in the cooking 
liquor (U.S. Environmental Protection Agency 1974). Consequently, heat- 
conditioned sludge displays a reduced affinity for water and an 
improved dewatering capacity (U.S. Environmental Protection Agency 1974, 
1978a). As shown in Figure 2-1 and described above, heat treatment 
also leads to the stabilization of sludge (U.S. Environmental Protec- 
tion Agency 1974, 1978b). Due to the high temperatures employed, all 
pathogens, including enteric viruses, in sludge are destroyed during 
heat conditioning (U.S. Environmental Protection Agency 1974, 1978b). 
Unfortunately, high energy costs often make heat conditioning of sludge 
impractical. 

Dewaterinq-dryinq beds . In the United States and Europe, 
sandbed drying (see Figure 2-1) is the most widely used method for 
sludge dewatering (U.S. Environmental Protection Agency 1974). Drying 



35 



beds consist of 6 to 9 inches (ca. 15 to 23 cm) of sand underlaid with 
approximately 12 inches (ca. 31 cm) of graded gravel or stone (U.S. 
Environmental Protection Agency 1974). Criteria for the design of 
drying beds can be found in the literature (U.S. Environmental Protec- 
tion Agency 1974, 1978a). In drying beds, water removal from sludge 
is accomplished first by drainage (filtrate is collected by underdrain 
system and is returned to the plant for further treatment) and then 
followed by evaporation (U.S. Environmental Protection Agency 1974). 
The effectiveness of sludge dewatering in drying beds is influenced by 
weather conditions (e.g., precipitation, solar radiation, air tempera- 
ture, and relative humidity), sludge characteristics (e.g., primary 
sludge dries faster than secondary sludge, digested sludge dries faster 
than raw sludge, and digested sludge dries faster than lime-stabilized 
sludge) and prior use of sludge conditioning (e.g., proper chemical 
conditioning can reduce sludge dewatering time by 50% or more) (U.S. 
Environmental Protection Agency 1974, 1978a). Sludge solids contents 
ranging from 45% for well -digested sludge to 90% for chemically 
conditioned sludge can be achieved on drying beds (U.S. Environmental 
Protection Agency 1974). As with other dewatering processes, sandbed 
drying reduces the volume of sludge to be further treated and disposed 
of (U.S. Environmental Protection Agency 1974). Such dewatering, 
therefore, reduces the fuel requirements of, for example, sludge 
incineration (U.S. Environmental Protection Agency 1974). 

During the air drying of sludge, it has also been demonstrated 
that enteric viruses are inactivated. Working with raw sludge (pH 6 or 



36 



less; lacking virucidal ammonia). Ward and Ashley (1977b) found a 
gradual reduction in the titer of seeded poliovirus type 1 (CHAT) 
during the air drying of sludge at 21 °C from 5% to 65% sludge solids 
content. However, when the sludge was allowed to dry to a solids 
content of 83% or greater, these investigators observed a dramatic 
decrease in poliovirus titer of greater than three orders of magni- 
tude in 4 days (similar results were also obtained using seeded cox- 
sackievirus Bl and reovirus 3). Ward and Ashley (1977b) went on to 
demonstrate that viral RNA is released during the air drying of sludge 
and this results in irreversible viral inactivation. Moreover, the 
evaporation process itself, and not some virucidal agent (note that 
the raw sludge used lacked the virucidal form of ammonia), was found 
responsible for poliovirus inactivation during the air drying of 
sludge at 21°C (Ward and Ashley 1977b). In fact, the inactivation 
rate of poliovirus incorporated into dewatered raw sludge was signifi- 
cantly lower than the inactivation rate of poliovirus seeded in raw 
sludge and allowed to air dry (Ward and Ashley 1977b). During heat 
treatment at 47°C or 51 °C, the inactivation rate of poliovirus type 1 
(CHAT) incorporated into dewatered raw sludge significantly declined as 
the sludge solids content was increased from 5% to 80% (Ward and Ashley 
1978b). Sludge solids content (or sludge moisture content) itself was 
shown to have an insignificant effect on the rate of poliovirus inac- 
tivation by heat (Ward and Ashley 1978b). Sludge solids-associated 
ionic detergents, however, were found to protect poliovirus in sludge 
from heat inactivation and to be concentrated during sludge dewatering 



37 



(Ward and Ashley 1978b). Hence, the greater protection from heat 
inactivation afforded to poliovirus as the sludge solids content was 
increased (Ward and Ashley 1978b). The ionic detergents in raw sludge 
were shown to be substantially degraded during the composting process 
and as a result, seeded poliovirus was heat-inactivated (39°C or 43°C) 
at a greater rate in composted sludge than in raw sludge held at the 
same sludge solids content (Ward and Ashley 1978b). Other enteroviruses 
(e.g., poliovirus 2, coxsackievirus A13 and coxsackievirus Bl ) have 
also been shown to be protected from heat inactivation in dewatered raw 
sludge (Ward and Ashley 1978b). 

From the research presented above, it can be concluded that 
substantial viral inactivation would occur, during the air drying of raw 
sludge at 21°C, only when sludge solids contents above 80% are achieved 
(Ward and Ashley 1977b). In actual practice, however, such sludge 
solids contents are rarely attained for raw sludge during sandbed drying 
(U.S. Environmental Protection Agency 1974). Furthermore, raw sludge is 
not routinely subjected to air drying because of the odors, insect 
pests, unsatisfactory drying rate and other problems associated with 
this practice (U.S. Environmental Protection Agency 1974). Thus, sand- 
bed drying is normally restricted to well-digested sludge (U.S. Environ- 
mental Protection Agency 1974). It can be hypothesized that entero- 
viruses are probably more rapidly inactivated in anaerobically digested 
sludge than in raw sludge when subjected to air drying. This is 
because anaerobically digested sludge usually contains virucidal 
ammonia (Ward and Ashley 1976, 1977a). This agent has been shown to 



38 



reverse the protective effect towards enteroviruses attributed to ionic 
detergents associated with sludge solids (Ward and Ashley 1977c, 1978a, 
1979; Ward et al_. 1976). Moreover, since the evaporation process 
itself was found primarily responsible for viral inactivation during 
the air drying of sludge (Ward and Ashley 1977b), and digested sludge 
has been shown to air dry at a more rapid rate and to a greater extent 
than raw sludge (U.S. Environmental Protection Agency 1974), it follows 
that viral inactivation would probably be greater in anaerobically 
digested sludge than in raw sludge. Ward and Ashley (1978b) also ob- 
served the inactivation rate of poliovirus type 1 (CHAT) seeded in 
dewatered raw sludge to increase with a corresponding increase in tem- 
perature. Consequently, viral inactivation is likely to be substan- 
tial ly accelerated during the air drying of sludge at higher temperatures 
than the 21 °C employed by Ward and Ashley (1977b). Under the most ideal 
conditions, however, sludge dewatering by air drying in sandbeds is not 
likely to yield a virus-free product. In Florida, for example, Wellings et 
al_. (1976a) detected 24 PFU of echovirus type 7 in 250 grams of air- 
dried (for 13 days) sludge obtained from drying beds. Due to the pos- 
sible viral hazard, all air-dried sludges should be handled with care 
during further treatment and final disposal (Wellings et al. 1976a). 
Dewaterinq-dryinq lagoons . Lagoons have also been commonly 
used in the United States for sludge dewatering (see Figure 2-1) (Fair 
et al. 1968; U.S. Environmental Protection Agency 1974). Bitton (1980b) 
reported that 264,000 dry tons of treated sludge (or 4.5% of the total 
sludge available) are discharged yearly into lagoons in the United 



39 



States. Due to the great potential for odor problems associated with 
sludge lagoons, only well -stabilized sludge has been recommended for 
dewatering in lagoons (U.S. Environmental Protection Agency 1974). 
Criteria for the design of drying lagoons can be found in the litera- 
ture (Sanks et al. 1976; U.S. Environmental Protection Agency 1974). 
Particularly important are the design criteria intended for the protec- 
tion of groundwater supplies. For example, the bottom of sludge lagoons 
must be at least 18 inches above the maximum groundwater table in order 
to prevent groundwater contamination (U.S. Environmental Protection 
Agency 1974). In drying lagoons , water removal from sludge is primarily 
achieved by evaporation (U.S. Environmental Protection Agency 1974). 
Therefore, the effectiveness of sludge dewatering in drying lagoons is 
mostly influenced by weather conditions (e.g., maximum drying rate in 
hot, arid climate) and by sludge depth (e.g., drying rate increases as 
the sludge depth decreases) (U.S. Environmental Protection Agency 1974). 
The little information available indicates that sludge dewatering in 
drying lagoons is an extremely slow process. Sludge held in a lagoon 
at depths of 2 to 4 feet, for example, required three years to dewater 
from 5% solids content to 45% solids content (U.S. Environmental Pro- 
tection Agency 1974) . 

Long-term lagooning has been found to destroy a significant 
fraction of the pathogens in digested sludge (U.S. Environmental Pro- 
tection Agency 1978b). In lagoons that had stopped receiving addi- 
tional quantities of digested sludge prior to their investigations, 
Sattar and Westwood (1979) and Farrah et al. (1981a) confirmed that 
indigenous enteric viruses associated with lagooned sludge are inac- 
tivated at a measurable rate. Under the warm temperatures of late 



->J 



40 



spring in Florida, Farrah et al. (1981a) found, for example, that 
the enterovirus titer of lagooned sludge dropped from 80 TCIDno/g 
of dry sludge to low or undetectable levels in approximately 6 weeks 
(note that similar decline was also observed for fecal coliforms in 
the lagooned sludge). In contrast, Sattar and Westwood (1979) detected 
enteric viruses in 39% of the sludge samples obtained from a lagoon 
in Ottawa, Canada, over a 14-month period (i.e., from April 1975 to 
May 1976). These investigators were able to recover viruses from sludge 
samples taken from the lagoon after 8 months. Clearly, indigenous 
viruses were inactivated at a much slower rate in the Canadian sludge 
lagoon (Sattar and Westwood 1979) than in the Floridian sludge lagoon 
(Farrah et al_. 1981a). Apparently, lower Ottawa temperatures (Sattar 
and Westwood 1979) contributed to greater viral persistence in the 
Canadian sludge lagoon. Whereas temperature appears to be an important 
factor affecting the inactivation rate of viruses in sludge lagoons, 
sludge drying is unlikely to have a significant effect. This is 
because, in lagoons, sludge dries at such a slow rate that substantial 
viral inactivation cannot be expected to result from the drying process 
itself. In addition to sludge treatment (i.e., drying and further di- 
gestion), lagoons also provide a temporary method of sludge storage 
(Fair et al. 1968; U.S. Environmental Protection Agency 1974). Ulti- 
mately, however, lagooned sludge must be disposed of and the method of 
choice is usually land application (U.S. Environmental Protection Agency 
1974). Although the research presented above indicates that long-term 



41 



lagooning substantially reduces the viral content of digested sludge, 
indigenous enteric viruses have, nevertheless, been routinely detected 
in lagooned sludge undergoing land application (Farrah et al^. 1981a; 
Sattar and Westwood 1979; Turk et al_. 1980). Consequently, all lagooned 
sludges should be handled with care during final disposal in order to 
avoid possible viral hazards (Sattar and Westwood 1979). 

Heat drying and reduction . Numerous heat drying and reduction 
processes (see Figure 2-1) are currently used for the removal of water 
from and for the reduction in the volume of sludge (U.S. Environmental 
Protection Agency 1974, 1978b). Reduction processes also destroy a 
major portion of sludge solids (U.S. Envrionmental Protection Agency 
1974). Due to the extremely high temperatures employed in these 
treatment processes, all pathogens, including enteric viruses, in 
sludge are destroyed (U.S. Environmental Protection Agency 1974, 1978b). 
Rising energy costs, however, are making these sludge treatment . 
processes impractical (U.S. Environmental Protection Agency 1974, 1978b). 
It should be pointed out that the heat-dried sludge or ash produced 
by the drying or reduction processes, respectively, require final dis- 
posal . 

Sludge irradiation . The treatment of sludge with ionizing 
radiation has recently been shown to be highly effective in destroying 
the pathogens, including enteric viruses, present in sludge (see 
review by Bitton 1980b). In particular, thermoradiation (i.e., ionizing 
radiation combined with moderate heat) has been demonstrated by Ward 
(1977) to rapidly inactivate poliovirus type 1 (CHAT) seeded in raw 



42 



sludge. The combined heat and radiation treatments appeared to have a 
synergistic effect on the survival of poliovirus in raw sludge (Ward 
1977). Due to its usual lack of pathogens, irradiated sludge has been 
reconmended for use as an animal feed supplement or for other agricul- 
tural purposes (Bitton 1980b). It is worth noting, however, that sludge 
irradiation is an energy-intensive process that has yet to become widely 
used in the treatment of sludge (note that it is not included in 
Figure 2-1 as a standard sludge treatment process). 

Final sludge disposal . Methods for final sludge disposal will 
be dealt with in a subsequent section. 

Viral and Other Health Hazards 
Associated with Treated Sludges 

As described above, most sludge treatment processes do not 
yield a virus-free product. With the possible exception of sludges 
treated at high temperatures, treated sludges represent potential health 
hazards due to the presence of viral pathogens (see review presented 
above). In addition to viruses, treated sludges (e.g., digested 
sludges) also contain bacterial pathogens (Foster and Engelbrecht 1973; 
Kowal and Pahren 1978), parasites (Hays 1977; Kowal and Pahren 1978; 
Little 1980; Pahren et al. 1979), toxic metals such as cadmium, copper, 
nickel, lead, zinc, and chromium (Chaney 1980; Jones and Lee 1978; Kowal 
and Pahren 1978; Pahren et al. 1979), and toxic organic residues such 
as aldrin, dieldrin, chlordane, heptachlor, lindane, toxaphene, poly- 
chlorinated biphenyls, and benzo(a)pyrene (Dacre 1980; Jones and Lee 1978; 
Kowal and Pahren 1978; Pahren et al. 1979). Clearly, the biological 



43 



and chemical properties of sludge are quite complex (Peterson et al_. 
1973; U.S. Environmental Protection Agency 1978b). Due to the numerous 
health hazards associated with treated sludges, final sludge disposal 
should be handled with the utmost of care. 

Final Sludge Disposal 

In 1979, municipal treatment plants in the United States were 
producing approximately 4.5 billion dry kg of sludge per year and 
this, is expected to rise to 3 billion by the early 1980s (Pahren et al. 
1979). Such large quantities of sludge coupled with the health hazards 
associated with sludge make final sludge disposal the most difficult 
of the sludge treatment processes (see Figure 2-1), Final sludge dis- 
posal is usually accomplished by either ocean dumping, sanitary landfill 
or land application (see Figure 2-1) (U.S. Environmental Protection 
Agency 1974, 1978b). Sludge incineration is not technically a final 
disposal method since ash is produced which requires disposal (in 
Figure 2-1, incineration is classified as a sludge reduction process). 
In actual practice, however, sludge incineration is considered a 
disposal method (Pahren 1980; Pahren et al. 1979; U.S. Environmental 
Protection Agency 1978b). 

Incineration . Of the total amount of sludge disposed of in 
1979 nationally, approximately 35% was incinerated (Pahren et al. 1979). 
In the future, however, sludge disposal by incineration is likely to 
be significantly curtailed due to air pollution, high energy costs and 
other problems associated with this disposal practice (Pahren 1980; 
Pahren et al. 1979; U.S. Environmental Protection Agency 1974, 1978b). 



44 



Moreover, sludge incineration destroys a potentially valuable resource 
which could be utilized, for example, on agricultural land (Pahren 
1980). 

Ocean dumping . For years, seacoast communities have been dis- 
charging digested sludge offshore in deep water (Fair et al^. 1968; 
U.S. Environmental Protection Agency 1974). Approximately 15% of the 
sludge disposed of in 1979 nationally was dumped in the ocean (Pahren 
et al^. 1979). By the end of 1981, however, ocean disposal of sludge 
will be prohibited by the U.S. federal government (Cowlishaw and Roland 
1973; Pahren 1980; Pahren et al_. 1979; U.S. Environmental Protection 
Agency 1974). The primary reason for prohibiting ocean dumping is that 
such a practice has been found to have long-term adverse effects on 
the ocean environment and on marine life (Cowlishaw and Roland 1973). 

Sanitary landfill . The burial of sludge (i.e., sludge covered 
by a soil depth greater than the plow layer) in a sanitary landfill 
is another popular and acceptable method for sludge disposal (U.S. 
Environmental Protection Agency 1974, 1978b). Of the total amount of 
sludge disposed of in 1979 nationally, approximately 25% was buried in 
sanitary landfills (Pahren et al_. 1979). In order to prevent odor, 
pathogen, operational and other problems, disposal in sanitary land- 
fills is usually restricted to well-stabilized, dewatered (^15% solids 
content for sludge-only landfills) sludge (U.S. Environmental Protec- 
tion Agency 1974, 1978b). The disposal of sludge in improperly 
managed landfills can result in groundwater pollution (Pahren 1980). 

Land application . Land application of sludge is receiving 
increased attention and will probably be the predominant sludge 



45 



disposal method of the future (U.S. Environmental Protection Agency 
1974, 1978b). Of the total amount of sludge disposed of in 1979 
nationally, approximately 25% was applied to land (Pahren et al_. 1979). 
In addition to achieving the goal of disposal, the application of 
sludge to strip-mined land and to cropland provides resource utiliza- 
tion in land reclamation and crop production, respectively (U.S. Environ- 
mental Protection Agency 1974, 1978a, 1978b). 

Sludge application benefits cropland in several ways. Nutrients 
which are abundantly present in municipal sludge are utilized effec- 
tively by growing plants (Cowlishaw and Roland 1973; Pahren 1980; Pahren 
et al_. 1979; U.S. Environmental Protection Agency 1974, 1978a, 1978b). 
Sludge application also improves several soil properties which are 
important for crop growth. For example, sludge addition increases the 
water content, water retention capacity, cation exchange capacity 
(CEC), organic carbon content and stable aggregate content of soils 
(Epstein 1975; Epstein et ai. 1976). The application of lime- 
stabilized sludge increases soil pH and can, therefore, increase sig- 
nificantly the productivity of acidic soils such as those found in many 
humid regions (Brady 1974). Land application of sludge involving 
resource utilization (e.g., cropland application) has distinct advan- 
tages over disposal -only methods and is favored by the U.S. Environmental 
Protection Agency (Pahren 1980; U.S. Environmental Protection Agency 
1978a). It has been estimated that only 1.3% of cultivated lands would 
be required for the application of all the sludge and animal waste 
produced in the United States (U.S. Environmental Protection Agency 
1978b). 



46 



The practice of applying sludge to land is not without its 
problems. Water movement, for example, has been found to be restricted 
(i.e., saturated hydraulic conductivity declined) in sludge- treated 
soils (Epstein 1975). Such a reduction in the soil conductivity has 
been attributed to the clogging of soil pores by microbial decomposition 
products (Epstein 1975). If improperly applied, sludge has been found 
to inhibit the growth of a previously planted crop. For example, lime- 
stabilized sludge was reported to form a filamentous mat on the soil 
surface which resulted in the partial inhibition of previously planted 
wheat (U.S. Environmental Protection Agency 1978a). No matting or 
crop inhibition was observed when lime-stabilized sludge was incorporated 
into the soil prior to planting (U.S. Environmental Protection Agency 
1978a). 

Undoubtedly, the major drawback of sludge application to land 
is the possible dissemination of pathogens and toxic chemicals leading 
to adverse effects on human and animal life (Burge and Marsh 1978; 
Elliott and Ellis 1977; Foster and Engelbrecht 1973; Kowal and Pahren 
1978; Pahren 1980; Pahren et al- 1979). In particular, enteric 
viruses present in sludge could potentially move through the soil 
matrix and contaminate groundwater supplies (see reviews by Berg 1973b; 
Bitton 1975; Bitton 1980a; Bitton et al. 1979b; Burge and Marsh 1978; 
Burge and Parr 1980; Cliver 1976; Duboise et al_. 1979; Elliott and 
Ellis 1977; Foster and Engelbrecht 1973; Gerba et al_. 1975; Moore et a^. 
1978; Sagik 1975). Due to the poor mixing and slow flow (generally 
<1 ft /day) conditions found in aquifers, many pollutants entering the 



47 



groundwater environment are not appreciably diluted and can persist 
for long periods of time (Lee 1976). 

Although sludge can be applied to land in several forms (i.e., 
liquid, dewatered or cake-dried), the application of sludge in the 
liquid form is usually preferred because of its simplicity (U.S. 
Environmental Protection Agency 1974, 1978b). For example, dewatering 
processes are not required and inexpensive transfer systems (e.g., 
tank trucks) can be employed for handling liquid sludges (U.S. Environ- 
mental Protection Agency 1978b). Liquid sludge is usually applied to 
land using one of the following methods: spray irrigation, surface 
spreading— ridge and furrow irrigation, surface spreading--fol lowed by 
sludge incorporation into the topsoil within 2 to 14 days, and subsurface 
injection (U.S. Environmental Protection Agency 1974, 1978b). There 
are potential problems associated with each of these application methods. 
While spray irrigation is a flexible method that requires minimum soil 
preparation, dangerous aerosols containing pathogens are generated during 
the spraying of sludge (U.S. Environmental Protection Agency 1974). 
The spreading of sludge on the soil surface can lead to the contamina- 
tion of surface waters via runoff and/or soil erosion (U.S. Environ- 
mental Protection Agency 1974). However, if the applied sludge is 
promptly incorporated into the topsoil, surface water pollution, odor 
and aesthetic problems are largely eliminated (U.S. Environmental Pro- 
tection Agency 1974). The injection of sludge below the soil surface 
avoids many of the problems associated with other application methods 
(e.g., aerosols, runoff and odors) (U.S. Environmental Protection Agency 



48 



1974). Due to the more favorable subsurface environment, however, 
pathogens (e.g., viruses) may persist longer in subsurface-injected 
sludge than in surface-applied sludge (Moore etal_. 1978). 

Whatever the application method employed, the disposal of 
sludge on land should be undertaken in accordance with local, state, 
and federal regulations and recommendations (Manson and Merritt 1975; 
U.S. Environmental Protection Agency 1974, 1978b; Wright 1975). 
Generally, only stabilized sludge is recommended for land application 
(U.S. Environmental Protection Agency 1974). Application rates of 
sludge to cropland vary depending upon sludge composition, soil charac- 
teristics, climate, vegetation and cropping practices (U.S. Environ- 
mental Protection Agency 1974), but should not exceed 20 dry tons of 
sludge solids/acre/year (44.8 dry metric tons/ha/year) or 46.8 m^/ha/ 
day (liquid rate) (Manson and Merritt 1975; U.S. Environmental Protec- 
tion Agency 1974). In order to prevent groundwater and surface water 
contamination, as well as other potential problems, Manson and Merritt 
(1975) recommended that sludge disposal sites conform to the following 
standards: 

1. High water tables should be no closer to the soil 
surface than 4 ft (1.22 m) 

2. Isolation from surface waters by a minimum distance 
of 200 ft (61 m) 

3. Maximum slope of 5% in order to prevent excessive 
surface runoff 

4. A crop that can be harvested is the preferred 
ground cover 



49 



5. A minimum distance of 250 ft (ca. 76 m) to the 
nearest residence 

6. Access to the sludge disposal site should be 
restricted 

Sludge disposal on properly managed sites in the United States has been 
successful and has not led to significant problems (Manson and Merritt 
1975; U.S. Environmental Protection Agency 1974). 

Fate of Sludge-Associated Viruses in Soils 

Literature pertaining to the survival and possible movement of 
enteric viruses in sludge-treated soils is presented in the Introductions 
to Chapters IV through VI. 



CHAPTER III 
EFFECT OF SLUDGE TYPE ON POLIOVIRUS 
ASSOCIATION WITH AND RECOVERY FROM SLUDGE SOLIDS 



Introduction 

The degree of association between viruses and sludge solids 
is a critically important factor in the assessment of the fate of 
these pathogens following sludge disposal on land. Yet, the nature of 
the association between viruses and sludge solids has not been adequately 
explored, partly because of the lack of virological methods. Recently, 
however, methods have been developed for the recovery of viruses from 
wastewater sludges. Practical methods for the recovery of viruses 
from sludge samples involve two steps. Because viruses in sludges 
have been found to be solids associated (Abid et a^. 1978; Glass et al. 
1978; Hurst et al_. 1978; Lund 1971; Ward and Ashley 1976; Wellings et 
al . 1976a), the first step of an effective method, therefore, consists 
of releasing both surface-adsorbed and solids-embedded viruses. The 
second step consists of concentrating the eluted viruses prior to viral 
assays. 

Various chemicals have been used to elute viruses from sludge 
solids, namely 0.1% sodium lauryl sulfate in 0.05 M glycine, pH 7.5 
(Abid et al_. 1978), tryptose phosphate broth (Moore et al^. 1978), 
3% beef extract, pH 9.0 (Wellings et al. 1976a), 3% beef extract, 
ambient pH (Glass et al_. 1978; Sattar and Westwood 1976), 0.05 M glycine 
buffer, pH 11.0 (Hurst et al,. 1978), 1% fetal calf serum in Earle's 

50 



51 



balanced salt solution, pH 9.5 (Subrahmanyan 1977), and 10% fetal 
calf serum (Sattar and Westwood 1976). Elution of solids-associated 
viruses may be aided by sonication (Abid et al^. 1978; Glass et al_. 
1978; Wei lings et al_. 1976a), shaking on a wrist-action shaker 
(Sattar and Westwood 1976), homogenization in a blender (Glass et al . 
1978; Moore et al_. 1978; Subrahmanyan 1977), or magnetic stirring 
(Abid et al_. 1978; Hurst et al_. 1978). Eluted viruses have been con- 
centrated by organic flocculation at low pH (Abid et al_. 1978; Glass 
etal. 1978; Hurst et al_. 1978), hydroextraction (Wellings et al_, 
1976a) or adsorption to bentonite clay (Turk et al_. 1980). 

Several of the methods proposed for the recovery of viruses 
from sludges do not contain a concentration step but simply involve 
the elution of viruses from sludge solids (Moore e_t al_. 1978; Sattar 
and Westwood 1976; Subrahmanyan 1977). The use of these methods is 
limited to raw sludges and other sludges containing large amounts of 
viruses. The concentration method proposed by Wellings et al_. (1976a) 
involving hydroextraction is cumbersome and requires considerably more 
time to perform than organic flocculation. The procedure developed 
by Abid et al_. (1978) is not practical, since it does not adequately 
concentrate the viruses eluted from sludge solids. Therefore, the 
best methods proposed to date for virus recovery from sludges appear to 
be those of Glass et al. (1978) and Hurst et al. (1978). Working with 
anaerobically digested sludge. Glass et al. (1978) obtained an overall 
recovery of poliovirus type 1 (CHAT) of 31%. Hurst et a^. (1978) found 
that poliovirus type 1 (LSc) could be recovered from activated sludge 



52 



samples with an overall efficiency of 80%. It is difficult to compare 
these two methods, since they were evaluated with different sludge 
types. 

The research reported in subsequent chapters of this disserta- 
tion deals primarily with the fate of enteroviruses following sludge 
disposal on land. In the course of conducting this research, the viral 
(i.e., indigenous and seeded) content of different sludge types had to 
be determined. Consequently, it was imperative to determine if viruses 
could be effectively recovered from the solids of different sludge types. 
The glycine method developed by Hurst et al. (1978) was evaluated for 
its effectiveness in recovering poliovirus type 1 (LSc) from different 
sludge types. The sludge types used were activated sludge mixed liquor, 
and anaerobically and aerobically digested sludges. It is the aim of 
this chapter to show that sludge type is a factor that can strongly 
influence the degree of association between viruses and sludge solids, 
as well as the recovery of sludge solids-associated viruses by the gly- 
cine method (Hurst et al^. 1978). 

Materials and Methods 
Virus and Viral Assays 

Poliovirus type 1 (strain LSc) was used in the research 
reported in this chapter. This virus strain is a non-neurotropic 
variant of the Mahoney strain, and is avirulent for mice and monkeys by 
all routes. This strain is identical to the virus designated as at- 
tenuated Sabin strain (Cooper 1967; Hahn 1972; World Health Organiza- 
tion 1968). Some general properties of polioviruses are shown in 



53 



Table 3-1. Stocks of the virus were prepared by infecting monolayer 
cultures of AV3 (a continuous line of human amnion), BGM (a continuous 
line of Buffalo green monkey kidney--Barron et aj[. 1970; Dahling et al. 
1974) or MA-104 (a continuous line of fetal rhesus monkey kidney) cells 
in a 32 oz (128 cm ) glass bottle (rubber-lined, screw-capped— Brockway 
Glass Co., Inc.). After allowing an adsorption period of 60 minutes 
with tilting at 15-minute intervals, the cells were overlaid with 40 ml 
of Eagle's minimal essential medium (MEM) supplemented with 10% fetal 
calf serum (FCS), 250 U/ml penicillin, and 125 yg/ml streptomycin (see 
Appendix for more details on the composition of this and other media 
used). After approximately 48 hours of incubation at 37°C, the overlay 
medium was decanted and then centrifuged at 270 x g for 15 minutes at 
4°C to remove cell debris. The resulting supernatant containing the 
virus was distributed in 1, 2 or 5 ml aliquots and immediately frozen 
at -70°C. The virus was kept at -70°C until used. 

Poliovirus was assayed by the plaque technique (Cooper 1967) on 
AV3, BGM or MA-104 cell monolayers prepared as follows. Confluent 
cell monolayers were grown in 32 oz glass bottles using Eagle's MEM 
supplemented with 10% FCS, 250 U/ml penicillin and 125 yg/ml strepto- 
mycin (i.e., growth medium; see Appendix). Each cell monolayer was 
then washed three times with 10 ml of the pre- trypsin solution (see 

Appendix). This treatment removes all traces of serum (contains 

+2 +2 
trypsin inhibitors), as well as Ca and Mg ions (enhance adsorption 

of cells to glass). To remove the cells from the glass bottle, 10 ml 

of the standard trypsin-versene solution (see Appendix) was then added 



54 



TABLE 3-1. General properties of polioviruses 



Property 



Value 



Nucleic acid 

Molecular weight of nucleic acid (daltons) 

Particle diameter (nm) 

Particle morphology 

Particle isoelectric point 

Stability at 25°C 

Stability at pH 3.0 

Stability in ether 



RNA^''^ (single-stranded) 

2 X 10^ 

27 to 30^'*^'^ 



Icosahedral 



a,b 



4.5 and 7.0" 
Relatively stable' 
Stable^'^'^ 
Stable^ 



From Davis et al^. (1973). 

^From Hahn (1972). 

^From Schwerdt and Schaffer (1955). 

From Mandel (1971), The data were obtained using poliovirus type 
1 (strain Brunhilde) . 



'From Bachrach and Schwerdt (1952) 



55 



and allowed to spread over the entire monolayer for 30 to 60 seconds. 
This solution was subsequently decanted and the cell culture was 
allowed to rest at room temperature until the cells came off the glass 
(approximately 5 minutes). Growth medium (10 ml) was then added, and 
pipetted twice up and down to dislodge the cells from the glass and 
break up clumps. An additional 190 ml of the growth medium was added 
to the content of the 32 oz glass bottle. This cell suspension was 
then distributed in 5 ml aliquots to 40 glass (2 oz--20 cm ) or plastic 
(25 cm ) bottles. After approximately 48 hours of incubation at 37°C, 
these small bottles contained confluent cell monolayers and were ready 
for use in viral assays. 

Experimental samples were diluted, if necessary, prior to assay 
in either Eagle's MEM containing 5% calf serum (rarely used) or 
phosphate-buffered saline (PBS) containing 2% PCS (both solutions 
contained 250 U/ml penicillin, 125 yg/ml streptomycin, and phenol red; 
see Appendix). All samples from each experiment were assayed on only 
one cell line (i.e., AV3, BGM or MA-104) using the procedure described 
below. A 0.6 ml aliquot of each diluted or undiluted sample was 
inoculated in fractions of 0.2 ml into three drained cell monolayers. 
Following inoculation, a 60-minute adsorption period with tilting at 
15-minute intervals was allowed. The infected monolayers were then 
overlaid with 4 ml of 1% methyl cellulose in Eagle's MEM supplemented 
with 5% PCS, 250 U/ml penicillin, 125 yg/ml streptomycin, and 117 yg/ml 
kanamycin (see Appendix). After incubation at 37°C for approximately 
48 hours, the cell monolayers were stained with either crystal violet 



56 



or neutral red (see Appendix). Plaques were subsequently counted with 
the unaided eye as suggested by Cooper (1967). In several experiments, 
the plaques were counted using an Omega photographic enlarger B22 
(Simmon Brothers, Woodside, New York). Each tabulated viral count 
represents the average of triplicate counts. The numbers of viruses were 
expressed as plaque-forming units (PFU). 



Sludges 

A variety of wastewater sludges was used in this study as listed 
in Table 3-2. The sludges were obtained from four wastewater treatment 
plants located in Gainesville and Pensacola, Florida. In addition, a 
lagooned sludge sampled at the West Florida Agricultural Experiment 
Station (Jay, Florida) was also used. The treatments the sludges 
received before being sampled are also shown in Table 3-2. One of the 
sludge types used was activated sludge mixed liquor. The term "mixed 
liquor" refers to the suspension undergoing treatment in an activated 
sludge unit. The abbreviated sludge designations (see Table 3-2) will 
be used to identify sludges in the rest of this dissertation. The 
sludges were collected in sterile Nalgene bottles, transported to the 
University of Florida (Gainesville) laboratory and then immediately 
refrigerated. All sludge samples were used within 30 days of sampling 
and most samples were used within 3 days. At the time of use, a 
sludge sample was first allowed to come to room temperature. The pH 
and solids content of the sludge was then determined. The pH was 
measured using a digital pH meter model 125 from Corning (Corning, New 
York). The solids content was determined by drying in an oven at 105°C 



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59 



for 24 hours a measured volume of sludge and was expressed as a per- 
centage on a weight (grams) to volume (milliliters) basis. Most of 
the sludges used were not autoclaved or decontaminated in any other 
way. Due to uncontrollable contamination of cell monolayers during 
viral assays, two sludge samples (GDAN and PDAN— see Table 3-2) had to 
be sterilized by autoclaving at 121°C with applied pressure of 15 psi 
for 15 minutes prior to use. During sludge treatment, digested sludges 
are frequently subjected to heat (i.e., 150 to 260°C under pressures of 
150 to 400 psi) as a conditioning step which improves subsequent 
sludge dewatering (U.S. Environmental Protection Agency 1978a; also 
see Figure 2-1). Thus, autoclaved sludge can be considered to be heat- 
conditioned sludge. As seen in Figure 3-1, the solids of autoclaved, 
anaerobically digested sludge (GDAN--see Table 3-2) settled in 2 
hours while those of nonautoclaved sludge remained dispersed. This 
increase in the rate of settling of sludge solids probably accounts for 
the improved-dewatering property of heat-conditioned sludge. Apart 
from the effect on the rate of settling of sludge solids, autoclaving 
probably did not significantly alter other sludge properties which 
affect sludge-virus interactions. For example. Ward and Ashley (1977a) 
found that autoclaving does not destroy the enterovirus-inactivating 
capacity of anaerobically digested sludge. Furthermore, autoclaving 
did not affect the degree of association between poliovirus and 
anaerobically digested sludge solids as determined below. 

Association of Seeded Poliovirus with Sludge Solids 

One milliliter of poliovirus stock in PBS containing 2% FCS was 
added directly to 100, 500, or 1,000 ml of sludge while stirring the 



FIGURE 3-1. Effect of autoclaving on the rate of settling of anaerobi- 
cally digested sludge solids 

An aliquot of anaerobically digested sludge (GDAN-- 
see Table 3-2; solids content, conductivity and pH 
equal to 2.0%, 3,250 ymho/cm at 25°C and 8.3, 
respectively) was autoclaved at 121°C with applied 
pressure of 15 psi for 15 minutes and compared to an 
aliquot of the sludge which had not been autoclaved. 
Approximately 21 ml each of autoclaved sludge 
and nonautoclaved sludge were added to graduated 
cylinders A and B, respectively. After 2 hours, 
the solids of the autoclaved sludge had settled 
while those of the nonautoclaved sludge remained 
dispersed. 



i1 




62 



suspension using either a magnetic stirrer or pipette (magnetic stirring 
could not be used for some sludges which had high solids contents). 
Poliovirus was seeded in the sludge samples at concentrations approxi- 
mately 1,000 times greater than the indigenous virus levels measured 
in the sludges used. Therefore, indigenous viruses present in the 
sludge samples did not affect the results presented herein. Magnetic 
stirring (or frequent mixing with a pipette) was continued for 10 
minutes to 60 minutes. Following the contact period, an aliquot of the 
unfractionated sludge (i.e., sludge sample without solids separated) 
was diluted in PBS containing 2% FCS and assayed directly for seeded 
viruses by the plaque technique. This assay was performed in order 
to determine if the direct viral assay after the contact period would 
agree with the calculated virus input based on the added volume of 
poliovirus stock of known titer. From Table 3-3, it can be seen that 
poliovirus was recovered with a mean efficiency of 109%, 104%, and ' 
98% : from mixed liquors, aerobically digested sludges and anaerobically 
digested sludges, respectively, following dilution and subsequent 
direct assay on cell cultures. Thus, poliovirus type 1 (LSc) added 
to sludge was recovered effectively after a contact period of 10 to 
60 minutes without any significant inactivation. Direct inoculation 
of unfractionated sludge into cell cultures has been found' to be toxic 
to cells (Nielsen and Lydholm 1980; Subrahmanyan 1977). In my research, 
the diluted sludge samples were not toxic to the cell cultures. Simi- 
larly, Hurst et al_. (1978) diluted virus-seeded activated sludge in 
Tris buffer and then successfully inoculated it directly into cell 
cultures without causing cell toxicity. Some of the sludges used in my 



"•'^T^'**';' 



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64 



study did, however, produce cell culture contamination when diluted and 
then directly inoculated into cell cultures. For these sludges (i.e., 
when not decontaminated by autoclaving) , the initial virus present was 
determined based on the amount of virus stock of known titer added to 
the sludge. From the results shown in Table 3-3, it is believed that 
the determination of the initial virus added to the sludge was accurately 
achieved by either direct viral assay of the sludge or based on the 
amount of virus stock of known titer added to the sludge. The total 
sludge volume was subsequently centrifuged at 1,400 x g for 10 minutes 
at 4°C (only an aliquot of sludges GDA180 and GDAN was centrifuged). 
An aliquot of the sludge supernatant produced was assayed for viruses. 
Thus, allowing the calculation of the "viable unadsorbed virus" frac- 
tion as shown below: 

viable unadsorbed ^ virus in sludge supernatant (total PFU) ,^- 
vi>"us (%) virus in unfractionated sludge (total PFU) ^ " 

(3-1) 

Furthermore, the "sludge solids-associated virus" fraction was also 
estimated as shown below: 

solids-associated virus (%) = 100 - viable unadsorbed virus (%) (3-2) 

For sludges GDA180 and GDAN, viral assays were performed, as described 
above (i.e., of unfractionated sludge and of sludge supernatant), several 
times throughout a 12-hour to 8-day period. During this period, the 
virus-seeded sludge was left at room temperature undisturbed, and was 
stirred only prior to obtaining a sample for viral assay. 



65 



Recovery of Seeded Poliovirus from Sludge Components 

The sludge supernatant and sludge solids generated were 
separately subjected to the virus recovery methodology described below. 
In several experiments, the sludge supernatants were not processed for 
virus concentration. These supernatants were simply adjusted to neutral 
pH and assayed for viruses as described previously. The number of 
viruses thus found were included in the "overall virus recovery" values 
reported. In those experiments in which the sludge supernatants were 
subjected to the virus concentration procedure, the supernatants were 
processed like the sludge solids eluates described below. 

The sludge solids-associated viruses were eluted and further 
concentrated using a modification of the glycine method developed by 
Hurst et al_. (1978). The solids were mixed with five volumes of 0.05 M 
glycine buffer, pH 11.5. The pH of the mixture was adjusted to between 
10.5 and 11.0 by the addition of 1 M glycine buffer, pH 11.5. The 
samples were vigorously mixed for 30 seconds using a magnetic stirrer 
and centrifuged at 1,400 x g f^r 5 minutes at 4°C (all centrifuga- 
tion was performed using a Sorvall RC5-B centrifuge, Ivan Sorvall 
Inc., Norwalk, Connecticut). The supernatants (i.e., the sludge solids 
eluates) were recovered, adjusted to neutral pH by the addition of 1 M 
glycine buffer, pH 2.0, and assayed for eluted viruses. The entire 
procedure described above was performed in less than 10. minutes. Thus, 
poliovirus was subjected to the high pH of 10.5 to 11.0 for no more 
than .10. minutes. Both Hurst etal. (1978) and Sobsey et al_. (1980b) 
observed no appreciable inactivation in .ID. minutes of poliovirus type 1 



66 



(LSc) seeded in 0.05 M glycine buffer, pH 10.5 to 11.0. Therefore, it 
is believed that there was no significant inactivation of poliovirus 
during the elution procedure. It should be noted, however, that this 
elution method is not practical for the recovery of reoviruses and 
rotaviruses from sludge. These virus types are rapidly inactivated 
when subjected to such high pH values (Sobsey et a^. 1980b). The 
viruses in the sludge solids eluates were concentrated by organic 
flocculation (Katzenelson et al_. 1976b) as follows. The eluates were 
adjusted to pH 3.5 by the addition of 1 M glycine buffer, pH 2.0, and 
the floes produced were pelleted by centrifugation at 1,400 x g for 
20 minutes at 4°C. The supernatants and pellets produced were treated 
separately. The supernatants were assayed for viruses and then passed 
through a series of 3.0, 0.45, and 0.25 ym Filterite filters (Filterite 
Corp., Timonium, Maryland) in a 47-mni holder. Adsorbed viruses were 
eluted from the filters with 7 ml of PBS containing 10% FCS, pH 9.0. 
The filter eluates were adjusted to neutral pH by the addition of 1 M 
glycine buffer, pH 2.0, and assayed for viruses. The filtrates (i.e., 
the fluids having passed the filters) were adjusted to neutral pH by 
the addition of 1 M glycine buffer, pH 11.5i,and assayed for viruses. 
The pellets previously obtained by centrifuging the samples at pH 3.5 
were mixed with five volumes of PBS containing 10% FCS, pH 9.0. The 
mixtures were adjusted to pH 9.0 by the addition of 1 M glycine buffer, 
pH 11.5, vortexed for 30 seconds and then centrifuged at 14,000 x g 
for 10 minutes at 4°C. The supernatants were adjusted to neutral pH 
by the addition of 1 M glycine buffer, pH 2.0,, and assayed for viruses. 



67 



Viral assays were performed at various steps in the procedure in order 
to determine the efficiency of the individual steps. Each "overall 
virus recovery" value reported was determined from the viruses recovered 
in the filter concentrate, pellet concentrate and sludge supernatant 
(concentrated or not). 

Statistical Treatment of Data 

Statistical treatment of the data was performed with the use of 
a Hewlett-Packard calculator model 9810A and Statistics Package V-6 
(Hewlett-Packard Company, Loveland, Colorado). 

Results and Discussion 

Association of Seeded Poliovirus 
with Sludge Solids 

Poliovirus seeded in the various sludge samples rapidly became 
associated with the sludge solids. However, no statistically signifi- 
cant linear correlation was found between the percent solids contents . 
of the sludges studied and the degree of virus association by the sludge 
solids (Table 3-4). This lack of correlation was found within each 
sludge type and for all sludge types combined. Thus, the sludge solids 
content was shown not to affect the association of virus with sludge 
solids, at least in the range of solids contents studied (i.e., 0.5% 
through 2.9%). This allowed sludges of different solids contents, but 
belonging to the same sludge type, to be grouped together in the same 
category. 

The mean percent of solids-associated viruses for activated 
sludge mixed liquors, anaerobically digested sludges, and aerobically 



68 






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70 



digested sludges was 57%, 70%, and 95%,. respectively (Table 3-4). 
Ward and Ashley (1976) have obtained similar results with anaerobically 
digested sludge. These investigators have shown that 65% and 67% of 
seeded poliovirus type 1 (Mahoney) and poliovirus type 1 (CHAT), 
respectively, were associated with anaerobic sludge solids after a con- 
tact period of 15 minutes. The association between seeded poliovirus 
and sludge solids was significantly greater for aerobically digested 
sludges than for mixed liquors or anaerobically digested sludges. No 
statistically significant difference was found between the mean 
percent of solids-associated viruses for mixed liquors and anaerobically 
digested sludges. The lagoon sludges mentioned in Table 3-4 are a 
mixture of aerobically digested sludge (1/3) and anaerobically digested 
sludge (2/3), and consequently, were placed in the category of 
anaerobically digested sludges. However, the association of poliovirus 
with lagoon sludge solids was greater than the association of this virus 
with other anaerobic sludge solids tested (see Table 3-4). Apparently, 
the presence of aerobic sludge solids (1/3) in lagoon sludge accounts 
for the greater ability of lagoon sludges to bind viruses. 

The reason for the greater association of seeded viruses with 
aerobic sludge solids is still unknown. However, aerobically digested 
sludges generally displayed lower pH values (ranging from 4.8 to 6.5) 
than mixed liquors (6.4 and 6.9) or anaerobically digested sludges 
(ranging from 6.4 to 8.3) (see Table 3-4). Virus adsorption to surfaces 
is promoted at low pH. Other parameters of aerobically digested sludges, 
as yet unidentified, could also account for the greater ability of these 



71 



sludges to bind viruses. However, we have shown that the sludge solids 
content does not account for the differences observed in the virus- 
binding capacity of different sludge types. In the range of sludge 
solids contents studied, there appears to be sufficient sites for virus 
binding and, therefore, this sludge parameter is not a limiting factor 
in determining the association of viruses with sludge solids. 

The degree of association between seeded poliovirus, and aerobic 
(see Table 3-5) or anaerobic (see Table 3-6) sludge solids remained 
fairly constant over a contact period of 8 days or 12 hours, 
respectively. Clearly, varying the contact time would not have sig- 
nificantly affected the results presented above on the virus-binding 
capacity of different sludge types. The inactivation rate of poliovirus 
seeded in sludge was also determined from the data shown in Tables 3-5 
and 3-6. In anaerobically digested sludge, the total amount of 
infectious poliovirus present in the sludge (i.e., virus in unfrac- 
tionated sludge) steadily declined over a 12-hour period (Table 3-6). 
In this sludge type, there was approximately a 50% reduction in the 
poliovirus titer in 12 hours (i.e., approximately 1 log,^ reduction/36 
hours) at room temperature (see Table-3-6). Ward and Ashley (1976) 
measured similar inactivation rates for poliovirus type 1 (CHAT and 
Mahoney) seeded in anaerobically digested sludge. In contrast to 
anaerobically digested sludge, there was no significant inactivation 
of poliovirus seeded in aerobically digested sludge during a 7-day 
contact period at room temperature (see Table 3-5). The low viral- 
inactivating capacity of aerobically digested sludge has also been 



72 



TABLE 3-5 


Effect of contact time on the 
poliovirus type 1 and aerobicc 


association between 

illy digested sludge solids 


Contact 
time 


Virus^ in 
unfractionated" 
sludge 
(total PFU) 


Virus 

in sludge 

supernatant^ 

(total PFU) 

4.2 X 10^ 


Viable 

unadsorbed" 

virus 

(%) 


Solids- 
associated^ 
virus 


30 min 


8.9 X 10^ 


0.5 


99.5 


60 min 


8.2 X 10^ 


4.8 X 10^ 


0.6 


99.4 


2 days 


1.2 X 10^ 


1.3 X lO'' 


1.1 


98.9 


4 days 


1.2 X 10^ 


4.3 X 10^ 


0.4 


99.6 


5 days 


8.0 X 10^ 


8.8 X 10^ 


1.1 


98.9 


6 days 


5.8 X 10^ 


1.9 X 10^ 


0.3 


99.7 


7 days 


8.2 X 10^ 


2.5 X 10^ 


0.3 


99.7 


8 days 


6.9 X 10^ 


2.5 X 10^ 


0.4 


99.6 



The virus was added to 1000 ml of aerobically digested sludge 
(GDA180— see Table 3-2; solids content and pH equal to 1.3% and 5.0, 
respectively) while stirring the suspension using a magnetic stirrer. 
Magnetic stirring was continued for 60 min. For the remainder of the 
experimental trial, the virus-seeded sludge was left at room temperature 
undisturbed, and was stirred only prior to obtaining a sample for viral 
assay. 

The sludge solids were not separated prior to assaying. 

The sludge was clarified by centrifugation at 1400 x g for 10 min 
at 4 C and the supernatant was subsequently assayed. 

The "viable unadsorbed virus (%)" values were calculated as shown 
in the Materials and Methods section. 

®The "solids-associated virus (%)" values were estimate d as shown 
in the Materials and Methods section. 



73 



TABLE 3-6, Effect of contact time on the association between poliovirus 
type 1 and anaerobically digested sludge solids 



Contact 

time 

(hours) 


Virus in 
unfractionated^ 
sludge 
(total PFU) 


Virus 
in sludge 
supernatant^ 
(total PFU) 


Viable 
unadsorbed" 
virus 

i%) 

41.8 


Sol ids - 

associated^ 

virus 

(%) 





1.1 X 10^ 


4.6 X 10^ 


58.2 


0.5 


1.1 X 10^ 


3.5 X 10^ 


31.8 


68.2 


1.0 


1.0 X 10^ 


4.4 X 10^ 


44.0 


56.0 


6.0 


9.4 X 10^ 


2.9 X 10^ 


30.9 


69.1 


8.5 


6.5 X 10^ 


2.3 X 10^ 


35.4 


64.6 


12.0 


' 5.3 X 10^ 


2.5 X 10^ 


47.2 


52.8 



The virus was added to 1000 ml of anaerobically digested sludge 
(GDAN— see Table 3-2> solids content and pH equal to 2.0% and 8.3, 
respectively) while stirring the suspension using a magnetic stirrer. 
Magnetic stirring was continued for 60 min. For the remainder of the 
experimental trial, the virus-seeded sludge was left at room temperature 
undisturbed, and was stirred only prior to obtaining a sample for viral 
assay. 

The sludge solids were not separated prior to assaying. 

The sludge was clarified by centrifugation at 1400 x g for 10 min 
at 4°C and the supernatant was subsequently assayed. 

^The "viable unadsorbed virus {%)" values were calculated as shown 
in the Materials and Methods section. 

The "solids-associated virus (%)" values were estimated as shown 
in the Materials and Methods section. 



74 



demonstrated in the field. Farrah et al. (1981a) found that aerobi- 
cally digested sludge contained larger indigenous viral titers than 
anaerobically digested sludge. The uncharged form of ammonia has 
been shown to exist in sludge (i.e.. tested raw and anaerobically 
digested sludge) mostly at pH values above 8 and to display virucidal 
activity against enteroviruses (Ward and Ashley 1976, 1977a). Due to 
the typically low pH of aerobically digested sludge (see Tables 3-4 
and 3-5), it can be hypothesized that the virucidal ammonia is probably 
largely absent from this sludge type. Therefore, the low viral- 
inactivating activity observed in the aerobically digested sludge 
(GDA180 sludge, pH 5.0) employed in this study (see Table 3-5) was to 
be expected. Similarly, Ward and Ashley (1976) found no appreciable 
inactivation of seeded poliovirus type 1 in .5 days . a.t: ZO^'C in raw 
sludge maintained at its naturally low pH of 6.0. Naturally high pH 
values, on the other hand, have been found in anaerobically digested 
sludge and consequently, this sludge type has demonstrated substantial 
viral -inactivating activity due to the presence of the virucidal 
ammonia (Ward and Ashley 1976, 1977a). The results presented in Table 
3-6 confirm that enteroviruses (i.e., used poliovirus type 1) seeded in 
anaerobically digested sludge (i.e., used GDAN sludge, pH 8.3) are 
inactivated at a significant rate. 

It is emphasized that the results and conclusions presented 
above pertain only to the virus used, poliovirus type 1 (LSc). Other 
enterovirus may, in fact, display different patterns of adsorption to 
sludge solids. Research has shown that only 20.7% of seeded echovirus 



75 



type 1 (Farouk) became associated with lagoon sludge solids after a 
contact period of 60 minutes (see Table 3-4). From Table 3-4 it can 
be seen that a larger fraction of poliovirus type 1 (83.9% and 91.1%) 
became associated with lagoon sludge solids. Goyal and Gerba (1979) 
have also shown that seeded echovirus type 1 (Farouk) does not adsorb 
well to a sandy loam soil. It is clearly established that virus type 
is a factor that affects virus adsorption to surfaces, including sludge 
solids. It was the aim of this chapter, however, to show that sludge 
type is also a critical factor influencing the degree of association of 
viruses with sludge solids. 



Recovery of Solids-Associated Viruses 

Seeded viruses that became associated with sludge solids were 
eluted and further concentrated according to the glycine method. In 
Table 3-7, it can be seen that significantly lower mean poliovirus 
recovery was found for aerobically digested sludges (15%) than for 
mixed liquors or anaerobically digested sludges (72%: and 60%^:., 
respectively). The mean poliovirus recoveries from mixed liquors and 
anaerobically digested sludges were not significantly different statis- 
tically. The recovery of solids-associated viruses was not dependent 
upon the volume of liquid sludge (100, 500, or 1,000 ml) processed. The 
mean poliovirus recovery from mixed liquors (72%): was similar to the 
recovery (80%) of the same virus reported by Hurst et al_. (1978). These 
researchers worked with activated sludge, which is the same sludge type 
as our mixed liquors. 

It is clearly established that the effectiveness of the glycine 
method in recovering solids-associated viruses is reduced for aerobically 



76 































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78 



digested sludges. The reason for the reduced recovery of viruses is 
that the elution step (i.e., mixing the sludge solids with five volumes 
of 0.05 M glycine buffer. pH 10.5 to 11.0, followed by rapid mixing for 
30 seconds) is not effective for this sludge type. For example, the 
mean elution of solids-associated viruses for this sludge type was only 
24%. In contrast, the mean elution of solids-associated viruses from 
mixed liquors and anaerobically digested sludges was 76% ,. and 80%, . 
respectively. The elution of viruses that was measured for mixed liquor 
solids (i.e.. 76%) ; closely approaches the value of 84% reported by 
Hurst etal. (1978) for the elution of poliovirus type 1 from activated 
sludge solids. The reason for the poor elution of solids-associated 
viruses from aerobic sludges has not been determined. However, this 
sludge type was able to bind a larger fraction of poliovirus than mixed 
liquors or anaerobically digested sludges (see Table 3-4). In order to 
understand the mechanism(s) involved, the chemical and physical nature 
of the solids of different sludge types should be studied. All other 
virus adsorption-elution steps of the glycine method (i.e., virus 
concentration steps) were equally effective in poliovirus recovery for 
all sludge types tested. 

Several methods have been proposed for the recovery of sludge 
solids-associated viruses and these methods are summarized in Table 3-8. 
Of the methods evaluated with enteroviruses seeded in sludge, all were 
tested for virus recovery efficiency using only one sludge type (see 
Table 3-8). The research presented above shows that sludge type in- 
fluences the recovery of poliovirus from sludge solids. Although the 



79 



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81 



results pertain only to poliovirus type 1 (LSc) and to the glycine 
method used to recover the solids-associated viruses, the indication 
is that sludge type is an important factor that should be considered 
when assessing the effectiveness of virus recovery methods. Research 
conducted using sludge artificially contaminated with virus has been 
questioned because it is believed that, unlike indigenous viruses, the 
seeded viruses become mostly adsorbed to the surface of sludge 
solids (Moore etal- 1977; Nielsen and Lydholm 1980). Indigenous 
viruses are believed to be mostly embedded within the sludge solids 
rather than merely surface adsorbed (Wellings et a^. 1976). In the 
case of wastewater-suspended solids, however, Stagg et al_. (1978) 
demonstrated that most (85%) of the indigenous, solids-associated 
col i phages were adsorbed to the surface of sewage solids rather than 
embedded. The exact nature of the association between seeded or 
indigenous viruses and sludge solids has not yet been conclusively 
determined. Whereas research performed with virus-seeded sludge may 
not completely simulate natural conditions, valuable information can, 
nevertheless, be obtained in less time and at lower cost than when 
working with indigenous viruses. Using seeded viruses, the research 
reported herein has elucidated the role of sludge type in the recovery 
of viruses from sludge solids. The results of this study lead one 
to suggest that future methods developed for the recovery of viruses 
from sludges be evaluated for the various sludge types likely to be 
tested. There is clearly a need for a method that can be shown to be 
effective in the recovery of viruses from a variety of sludge types. 



CHAPTER IV 

POLIOVIRUS TRANSPORT STUDIES INVOLVING 

SOIL CORES TREATED WITH VIRUS-SEEDED SLUDGE 

UNDER LABORATORY CONDITIONS 



Introduction 
The application of wastewater sludge to land is receiving 
increased attention and will probably be the predominant sludge dis- 
posal method of the future (U.S. Environmental Protection Agency 1974, 
1978b). There is some concern, however, that this practice may result 
in the contamination of groundwater supplies with pathogenic viruses 
(see reviews by Berg 1973b; Bitton 1975, 1980a; Bitton et al. 1979b; 
Burge and Marsh 1978; Burge and Parr 1980; Oliver 1976; Duboise et 
ai- 1979; Elliott and Ellis 1977; Foster and Engelbrecht 1973; 
Gerba et al_. 1975; Moore et al. 1978; Sagik 1975). Unfortunately, 
the transport pattern (i.e., movement or retention) of sludge- 
associated viruses in soils has not been adequately evaluated. The few 
studies that have been conducted indicate that enteroviruses seeded in 
anaerobically digested sludge are effectively retained by the soil 
matrix (Damgaard-Larsen et al. 1977; Moore et al. 1978; Sagik 1975). 
Since both seeded and indigenous viruses have been found to be associated 
with the sludge solids (Abid et al. 1978; Glass et a^. 1978; Hurst et al. 
1978; Lund 1971; Ward and Ashley 1976; Wei lings et al. 1976a; also see 
Table 3-4), it follows that viruses are probably immobilized along with 
the sludge solids in the top portion of the soil profile (i.e., during 
surface spreading of sludge) or at the injection site within the soil 

82 



83 



matrix (i.e., during subsurface injection of sludge) (Cliver 1976). 
Moreover, it appears that viruses are not readily dissociated from sludge 
solids in the soil environment (Burge and Parr 1980; Sagik 1975). 

In this chapter, results of poliovirus (type 1, strain LSc) 
transport studies involving soil cores treated with virus-seeded sludge 
are presented. These studies were conducted under controlled laboratory 
conditions. The effect of sludge type (used anaerobically digested 
sludge, conditioned-dewatered sludge, chemical sludge and lime-stabilized, 
chemical sludge), soil type (used a Red Bay sandy loam and a Eustis fine 
sand), soil core type (used laboratory-packed soil columns and undis- 
turbed soil cores) and application regime (virus-seeded sludge was 
applied continuously or in a spiked fashion) on the transport of sludge- 
associated poliovirus in soil cores was evaluated. The capacity of rain 
water to elute poliovirus from the sludge-soil matrix was also investi- 
gated. All soil cores were leached under saturated flow conditions. 
The information gained from this study should shed further light on the 
role of mineral soils in retaining sludge-associated viruses. 

Materials and Methods 
Virus and Viral Assays 

Poliovirus type 1 (strain LSc) was used in the research reported 
in this chapter. Some general properties of polioviruses are shown in 
Table 3-1. Stocks of the virus were prepared as described in Chapter 
III (see page 53). The virus was kept at -70°C until used. Poliovirus 
was assayed by the plaque technique as described in Chapter III (see 
pages 53-56). Each viral count shown represents the average of 



84 



triplicate counts. The numbers of viruses were expressed as plaque- 
forming units (PFU). 

Primary Wastewater Effluent 

Primary wastewater effluent was obtained from the University 
of Florida campus wastewater treatment plant, Gainesville, Florida. The 
detention time of raw wastewater in the primary settlers was approxin 
mately 2 hours. No chlorine residual was found in the primary effluent 
sample used (i.e., by the orthotolidine test). The sample of primary 
effluent used was collected, and its pH and conductivity were measured 
as described below for digested sludges. 

Sludges 

Several sludge types were used in the research reported in this 
chapter and they are described below. 

Digested sludges . Anaerobically digested sludges sampled at 
the Main Street wastewater treatment plants of Gainesville and Pensacola, 
Florida (6DAN and PDAN, respectively— see Table 3-2), were used. The 
sludges were collected and sludge parameters (i.e., pH and solids con- 
tent) were measured as described in Chapter III (see page 56). The 
sludge conductivity was measured using a Beckman conductivity bridge 
model RC 16B2 (Beckman Instruments, Fullerton, California). Due to 
uncontrollable contamination of cell cultures during viral assays, these 
sludge samples had to be sterilized by autoclaving at 12rc with applied 
pressure of 15 psi for 15 minutes prior to use. As explained earlier 
(see page 59), autoclaving did not significantly affect the sludge-virus 



85 



interactions and the autoclaved sludge can simply be considered to be 
heat-conditioned. The sludges were used undiluted or diluted (1:50, 
vol. /vol.) with either distilled water or 0.01 N calcium chloride. 
The pH and conductivity of each diluted sludge sample was also 
measured as described above for undiluted sludge. 

Sludge liquor . Anaerobically digested sludge liquor was pro- 
duced by centrifuging (all centrifugation was performed using a Sorvall 
RC5-B centrifuge, Ivan Sorvall Inc., Norwalk, Connecticut) GDAN sludge 
(see above) at 14,000 x g for 10 minutes at 4°C. This procedure was 
performed again on the decanted supernatant and this yielded the clear 
sludge liquor. Chemical parameters for this sludge liquor were deter- 
mined by the Analytical Research Laboratory, Soil Science Department, 
University of Florida, Gainesville, and are presented in Table 4-1. 
The sludge liquor (containing 0.01 N calcium chloride) was also used 
to dilute (1:50, vol. /vol.) the GDAN sludge. Lagoon sludge (LAG— see 
Table 3-2; a mixture of 1/3 aerobically digested sludge and 2/3 
anaerobically digested sludge) liquor was also used and it was produced 
by the centrifugation of lagoon sludge as outlined above for GDAN 
sludge. The lagoon sludge liquor was passed through a series of 
0.45- and 0.25-ym Filterite filters (Filterite Corp., Timonium, Maryland) 
in a 47-mm holder and then adjusted to pH 8.0 using 0.01 N NaOH prior 
to use. The pH and conductivity of each sludge liquor sample was 
measured as described above for undiluted digested sludges. 

Condi tioned-dewatered sludge . Poliovirus-seeded, GDAN sludge 
(see above) was conditioned with 1200 mg/£ of the cationic polymer, 



86 



TABLE 4-1 , 



Chemical parameters for the anaerobically digested sludge 
liquor 



Parameter^ 


Sludge*^ liquor 
value 
(ppm) 


Parameter^ 


Sludge liquor 
value 
(ppm) 


Soluble salts 


703 


As 





Na 


63 


Cd 





K 


29 


Cr 





Ca 


20 


Cu 


0.05 


Mg 


15 


Ni 





Al 





Pb 





Fe 


1.05 


Zn 


0.04 



Chemical parameters were determined by the Analytical Research 
Laboratory, Soil Science Department, University of Florida, Gainesville. 

Anaerobically digested sludge (GDAN— see Table 3-2 ; solids con- 
tent, conductivity and pH equal to 2.0%, 3250 ymho/cm at 25°C and 8.3, 
respectively) was centrifuged at 14,000 x g for 10 min at 4°C. This 
procedure was performed again on the decanted supernatant and this 
yielded a clear sludge liquor. 



87 



Hercufloc #871 (Hercules Co., Atlanta, Georgia). The polymer was added 
to 1000 ml of the virus-seeded sludge while mixing rapidly on a mag- 
netic stirrer. Mixing was continued slowly for an additional 5 minutes. 
The entire sludge sample was then centrifuged at 320 x g (i.e. , 1400 
rpm) for 10 minutes at 25°C. The supernatant was decanted, assayed for 
viruses, and discarded. The dewatered sludge volume and sludge solids 
content (i.e., as percent, by method described above for digested 
sludges) was measured. The conditioned-dewatered sludge produced 
(i.e., in duplicate) was then assayed for viruses as described below. 
The procedure employed above in the conditioning and dewatering of the 
sludge was identical to that used at the Main Street wastewater treat- 
ment plant, Gainesville, Florida (Dr. DuBose, Main Street plant, per- 
sonal communication; see Figure 6-T). 

Chemical sludges . The chemical sludges were precipitated from 
1000 ml of poliovirus-seeded, raw sewage using the general procedure 
of Sattar et al_. (1976) and the coagulants, alum (i.e., aluminum sul- 
fate),. ferric chloride or lime (i.e., calcium hydroxide). The raw sewage 
used was obtained from the University of Florida campus wastewater treat- 
ment plant or from the Main Street wastewater treatment plant, both being 
located in Gainesville, Florida. The raw sewage samples were collected 
and sewage parameters (i.e., pH and conductivity) were measured as 
described above for sludge samples. The raw sewage samples were sterilized 
by autoclaving at 121°C with applied pressure of 15 psi for 15 minutes 
prior to use. Viral assays were made before and after the addition 
of coagulant. The final concentration in sewage of alum was 300 mg/i 



88 



[as Al2(S04)3-18 H2O] and of ferric chloride was 50 mg/£ (as FeCl ). 
Similar concentrations of these coagulants were used by Wolf et al_. 
(1974) in the precipitation of an activated sludge effluent as a 
tertiary treatment process. Following the addition of alum or ferric 
chloride, the pH of the solution was adjusted using 0.2 N HCl to 6.0 or 
5.0, respectively, in order to achieve maximum flocculation (Fair et al. 
1968). The coagulant, lime, was added until a pH of 11.1 to 11.3 [i.e., 
final concentration in sewage of 150 to 250 mg/£ of Ca(0H)2] was 
achieved (according to Sattar et a],. 1976). Following the addition 
of the coagulants, the sewage samples were mixed on a magnetic stirrer 
rapidly for 10 minutes and slowly for 5 minutes. The flocculated 
sewage samples were then transferred to Imhoff cones and 60 minutes 
was allowed for the formation and settling of the chemical sludges 
(see Figure 4-1). The supernatants in the Imhoff cones were assayed 
for viruses and discarded. The sludge volume and sludge solids content 
(i.e., as percent, by method described above for digested sludges) was 
measured for each chemical sludge. The chemical sludges produced (i.e., 
in duplicate for the lime sludge, and in triplicate for alum and ferric 
chloride sludges) were then assayed for viruses as described below. 
Lime-stabilized, chemic al sludges . A sample of alum sludge 
and a sample of ferric chloride sludge, produced and assayed for viruses 
as described above, were lime-stabilized according to the procedure of 
Farrell et al. (1974). The sludges were treated with an aqueous slurry 
of lime [5% (wt./vol.) stock of Ca(0H)2] ""^^'^ ^ P" 0^ 11-5 was achieved 
and maintained for 5 minutes. The final concentration of lime [as 



FIGURE 4-1. Chemical sludges (i.e., 
from poliovirus-seeded, 
settling for 60 minutes 



lime and alum) were precipitated 
raw sewage and are shown after 
in Imhoff cones 



— — '■ — ■ — ■ " -•.'•^ vr-- -' y 

91 



Ca(0H)2] added to the alum and ferric chloride sludge was 1389 and 625 
mg/£, respectively. A contact time of 30 minutes was allowed while 
mixing the suspension on a magnetic stirrer. After 30 minutes of mixing, 
the pHs of the sludges were measured and had dropped to 11.1 or 11.3. 
The lime-stabilized, chemical sludges were then assayed for viruses as 
described below. 

Association of Seeded Poliovirus with Sludge Solids 

Poliovirus stock in phosphate-buffered saline (PBS) containing 
2% fetal calf serum (FCS) (see Appendix for more details on the compo- 
sition of this solution) was added directly to 0.01 N calcium chloride, 
primary wastewater effluent, anaerobically digested sludge (diluted or 
undiluted) and sludge liquor at the rate of 1 ml of virus stock per 
1000 ml of solution and while stirring the suspension using a magnetic 
stirrer (see Chapter III, page 59). After a 1-minute mixing period, 
an aliquot of the samples containing low solids contents (i.e., 0.01 N 
calcium chloride, primary wastewater effluent, diluted anaerobically 
digested sludge, and sludge liquor) was diluted in PBS containing 2% 
FCS and assayed directly for seeded viruses by the plaque technique. 
This viral assay was performed in order to determine the amount of 
virus present in these samples initially and was repeated at the end 
of experimental trials in order to assess any viral inactivation which 
might have occurred. No attempt was made to determine the degree of 
association between seeded poliovirus and the small quantity of solids 
present in these samples. 

The association of poliovirus with sludge solids was deter- 
mined for undiluted anaerobically digested sludge (seeded with 



92 



poliovirus and suspension magnetically mixed for 10 to 60 minutes), 
conditioned-dewatered sludge (poliovirus transferred to this sludge 
during dewatering process), chemical sludges (poliovirus transferred to 
chemical sludges during precipitation of virus-seeded, raw sewage) and 
lime-stabilized, chemical sludges. The procedures used are outlined 
in detail in Chapter III (see page 62). Briefly, an aliquot of the 
unfractionated sludge (i.e., sludge sample without solids separated) 
was diluted in PBS containing 2% FCS and assayed directly for viruses by 
the plaque technique. This method (i.e., sludge dilution and subsequent 
direct assay on cell cultures) has been previously shown to be highly 
efficient in the recovery of poliovirus from unfractionated sludge 
(see Chapter III, page 62 and Table 3-3). The unfractionated sludge 
assay was performed in order to determine the total amount of virus 
present in the sludge sample. An aliquot of the sludge was subsequently 
centrifuged at 1400 x g for 10 minutes at 4°C. The sludge supernatant 
produced was assayed for viruses. The "viable unadsorbed virus" and 
"sludge solids-associated virus" fractions were calculated as shown 
in Chapter III (see page 64). .. 

Rain Water 

Rain water was collected next to the Environmental Engineering 
Sciences building at the University of Florida, Gainesville. Chemical 
parameters for the rain water used were determined by Hendry (1977) 
and are presented in Table 4-2. The rain water was sterilized by auto- 
claving at 121°C with applied pressure of 15 psi for 15 minutes prior 
to use. 



93 



TABLE 4-2. Chemical parameters for the rain water used in this study 



Parameter 

pH 

Conductance 

TOC 

TKN 

nhJ-n 

NO3-N 
Ortho-P 
Total -P 
Na 



Value' 



4.46 
23.9 
5.20 
0.69 
0.10 
0.17 
0.016 
0.032 
0.33 



Parameter 


Value' 


K 


0.15 


Ca 


0.50 


Mg 


0.07 


CI" 


0.71 


504"^ 


1.84 


Cd 


5.7 


Pb 


15.2 


Cu 


39.4 


Zn 


28.2 



^Data were adapted from Hendry (1977). The values shown repre- 
sent average weighted concentration of individual rain events col- 
lected next to the Environmental Engineering Sciences building. 
University of Florida, Gainesville, from June 1976 to May 1977. 

"Value in ymho/cm. The values for Cd, Pb, Cu, and Zn are in 
ug/1. All other values except pH are in mg/1 . 



94 



Soils 

The soils studied were a Red Bay sandy loam sampled at the West 
Florida Agricultural Experiment Station, Jay, and a Eustis fine sand 
sampled at the agronomy farm, University of Florida, Gainesville. The 
Red Bay sandy loam has been classified as a Rhodic Paleudult, fine- 
loamy, siliceous, thermic while the Eustis fine sand was classified as a 
Psammentic Paleudult, sandy, siliceous, hyperthermic (Calhoun et al . 
1974). Some characteristics of these soils are shown in Table 4-3. 
The percent organic matter in these two soils was measured at less 
than ]% except for the Al horizon of the Red Bay sandy loam which was 
found to contain 4.3% organic matter (Calhoun et al. 1974). 

Poliovirus Transport Studies 

Poliovirus transport (i.e., movement or retention) in soil cores 
treated with virus-seeded sludge was studied under laboratory conditions. 
Two types of soil cores were used as described below. 

Laboratory-packed soil columns . Laboratory-packed soil columns 
were prepared using subsoil samples of Red Bay sandy loam (consisted 
mainly of the A2 and Bit horizons— see Table 4-3) and Eustis fine sand 
(consisted mainly of the A21 and A22 horizons— see Table 4-3). Each 
subsoil sample was screened by hand to remove rocks and large organic 
matter, and was then allowed to air dry. The soils were not autoclaved 
or sterilized in any other way. The dry soils were then carefully 
packed into acrylic plastic columns 10 cm or 29 cm in length (packed 
10 cm or 27 cm with soil, respectively) and 4.8-cm internal diameter. 
A polypropylene screen (105-ym pore size) was used to support the soil 



95 



TABLE 4-3. Some characteristics of the soils under study 



Soil^ 


Soil 
horizon 


Depth 


Description 


Mechanical composition (%) 




(cmj 


















Sand 


Silt 


Clay 










(2- 


(0.05- 


(< 0.002 mm) 










0.05 mm) 


0.002 mm) 




Red 


Al 


0-15 


Dark brown 


66.0 


20.4 


13.6 


Bay 






fine sandy loam 








sandy 














loam^ 


A2 


15-30 


Yellowish-red 
sandy loam 


61.0 


20.0 


19.0 




Bit 


30-48 


Red sandy 
clay loam 


56.0 


15.4 


28.6 




B21t 


48-97 


Red light 
sandy clay 


52.4 


11.4 


36.2 


Eustis 


Ap 


0-25 


Dark gray 


94.8 


2.4 


2.8 


fine 






fine sand 








sand*^ 
















A21 


25-58 


Light yellowish- 
brown fine sand 


94.4 


2.0 


3.6 




A22 


58-102 


Light yellowish- 
brown fine sand 


94.3 


2.3 


3.4 




A23 


102-135 


Light yellowish- 
brown fine sand 


94.7 


1.6 


3.7 




A24 


135-163 


Yellowish-brown 
fine sand 


93.9 


1.3 


4.8 



^Adapted from Calhoun et al. (1974). 

Identification by x-ray diffraction. 

'^Sample was taken in Santa Rosa County, Florida; West Florida 
Agricultural Experiment Station, Jay. 

Sample was taken in Alachua County, Florida; agronomy farm. 
University of Florida, Gainesville. 



96 



TABLE 4-3. 


Extended. 






Dominant 
clay^ 


pH 
(in 1:1 
water) 


Bulk density 
(g/cm3) 


Saturated 

hydraulic 

conductivity 

(cm/hr) 


Vermiculite 


5.3 


1.26 


12.1 


Vermiculite 


5.4 


1.43 


15.9 


Vermiculite 


5.6 


1.48 


20.2 


Gibbsite 


5.1 


1.60 


21.6 


— 


6.7 


1.62 


28.0 


— 


6.5 


1.59 


27.2 


— 


6.5 


1.54 


34.0 


— 


6.3 


1.55 


52.6 


— 


6.0 


1.53 


54.2 



97 



in each column while allowing the free movement of viruses (i.e., 
did not adsorb viruses in soil leachates). The columns were packed 
uniformly at a constant rate while gently tapping to prevent soil 

subsidence (Drewry and Eliassen 1968). The field bulk densities of 

3 
1.45 g/cm for the Red Bay sandy loam (i.e., average for the A2 and 

3 
Bit horizons— see Table 4-3) and 1.56 g/cm for the Eustis fine sand 

(i.e., average for the A21 and A22 horizons— see Table 4-3) were repro- 
duced in the soil columns by packing the appropriate grams of dry soil 

3 
in the measured volume of each column (i.e., 180.86 cm for the 10-cm 

3 
column and 488,33 cm for the 27-cm column). The soil columns were 

then placed in soil column holders (supplied by Soil Moisture Equipment 
Corp., Santa Barbara, California) as shov/n in Figures 4-2 and 4-3 for 
10-cm and 27-cm columns, respectively. The air in the columns was then 
displaced by flushing with carbon dioxide for approximately 60 minutes 
in order to ensure subsequent uniform wetting of the soil. The soil 
columns were then conditioned by passing 2 to 5 pore volumes of non- 
seeded 0.01 N calcium chloride, distilled water, rain water, or sludge 
liquor using a peristaltic pump (Buchler, Fort Lee, New Jersey). The 
conditioning solution used was identical or similar to the test 
solution. For example, soil columns ultimately receiving virus-seeded, 
anaerobically digested sludge diluted (1:50, vol. /vol.) with 0.01 N 
calcium chloride were previously conditioned with 0.01 N calcium 
chloride. For soil columns receiving undiluted sludge (e.g., chemical 
sludge), rain water was used as the conditioning solution. Following 
conditioning, poliovirus was suspended in 0.01 N calcium chloride, sludge 






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102 



liquor or diluted anaerobically digested sludge and was subsequently 
applied continuously to soil columns at approximately 5 ml/min using 
the peristaltic pump. As detailed above, influent poliovirus concen- 
tration was determined from viral assays made at the beginning and end 
of each column experiment. In several experiments, a shift was made to 
the application of nonseeded 0.01 N calcium chloride or rain water con- 
tinuously (at approximately 5 ml/min) by the peristaltic pump in order 
to determine if these solutions could elute adsorbed viruses. The 
undiluted sludge samples (i.e., conditioned-dewatered sludge, chemical 
sludges and lime-stabilized, chemical sludges) could not be applied to 
the soil columns via the peristaltic pump because of the high solids 
contents. These sludges were applied directly on top of the soil 
columns, were allowed to soak in, and then, were worked under 2.5 cm. 
The poliovirus concentrations in these sludges was determined as 
described earlier. Following the application of the sludges, the 
covers on the soil columns (i.e., the top plates of the soil column 
holders) were replaced and the soil columns were then leached with non- 
seeded rain water. The rain water was applied continuously to the 
soil columns at approximately 5 ml/min using the peristaltic pump. 
After percolation through the soil, the leachates from all laboratory- 
packed columns were collected in sterile screw-capped bottles and 
assayed for viral infectivity as described below. 

Undisturbed soil cores . Undisturbed soil cores (Blake 1965; 
Sanks et al^. 1976) were also used in poliovirus transport studies. 
Undisturbed cores were obtained in driving polyvinyl chloride pipes 



103 



into the soil to the desired depth according to the procedure of Blake 
(1965). Care was taken not to compress the soil in the pipes during 
sampling, and, thereby, to preserve the natural structure and packing 
of the soil as nearly as possible in the pipes. This was accomplished 
by ensuring that the elevation of the soil inside the pipes was the same 
as the elevation of the surface soil outside the pipes during sampling 
(Blake 1965). The soil around the pipes was then removed with a shovel 
and the soil cores were gently removed. In the laboratory, soil ex- 
tending beyond the bottom end of each core was trimmed with a spatula. 
A polypropylene screen (105-ym pore size—see above) and a spout were 
secured at the bottom of each undisturbed soil core. Red Bay sandy 
loam undisturbed cores were sampled at the West Florida Agricultural 
Experiment Station at Jay. These soil cores were 54 cm in length 
(pipes were 60 cm in length) and had an internal diameter of 5.0 cm, 
and, thereby, consisted of the Al , A2, Bit and B21t horizons of the sandy 
loam (see Table 4-3), The Red Bay sandy loam cores were not conditioned 
prior to use. Eustis fine sand undisturbed cores were sampled at 
the agronomy farm. University of Florida, Gainesville. These soil 
cores were 33 cm in length (pipes were 40 cm in length) and had an in- 
ternal diameter of 5.0 cm, and thereby, consisted of the Ap and A21 hori- 
zons of the fine sand (see Table 4-3). The Eustis fine sand cores were 
conditioned with either 5 pore volumes of rain water or 0.01 N calcium 
chloride. All undisturbed soil cores were then treated with undiluted 
anaerobically digested sludge (GDAN— see above) which had been seeded 
with poliovirus. The poliovirus concentrations in the anaerobically 



104 



digested sludge samples and the degree of poliovirus association with 
the sludge solids were determined as described earlier in this chapter. 
Following viral assays, one inch or 2.5 cm (51.6 ml) of poliovirus- 
seeded sludge was applied to each soil core, allowed to soak in and 
then was worked under 2.5 cm. In one experiment with two Red Bay sandy 
loam cores, the applied sludge was allowed to air dry for 24 hours 
(the cores were placed on the roof of the Environmental Engineering 
Sciences building. University of Florida, Gainesville) before being 
worked under 2.5 cm. Following incorporation of the sludge solids into 
the top inch of soil, the soil cores were eluted with either 0.01 N 
calcium chloride or rain water. These solutions were applied from 
inverted, self-regulated, 1-liter Erlenmeyer flasks set to maintain 
a 2.5-cm hydraulic head on the cores (Sanks et al^. 1976). After perco- 
lation through the soil, leachates from the undisturbed soil cores were 
collected in sterile screw-capped bottles and assayed for viral 
infectivity as described below. The percolation rate of fluid through 
each undisturbed soil core was determined by measuring the time required 
to collect a known volume of leachate. 

Leachates from transport studies . The leachates from 
laboratory-packed soil columns and undisturbed soil cores were collected 
in pore volumes or fractions of pore volumes. The pore volume of each 
soil core (or column) is the volume within the core which is not 
occupied by the soil particles (Brady 1974) and is determined by first 
calculating the percent pore space of the soil as shown below: 



pore space (%) = 100 - 



bulk density ^ ^qq 



particle density 



(4-1) 



105 



The bulk density is defined as the mass of dry soil in a unit volume 
consisting of soil solids and pores (Blake 1965; Brady 1974). The 
particle density is defined as the mass of a unit volume of soil solids 
(Brady 1974). The bulk density of the Red Bay sandy loam was 1.45 g/cm 

(used this value for both laboratory-packed columns and undisturbed 

3 
cores), and that of the Eustis fine sand was 1.56 or 1.61 g/cm (for 

laboratory-packed columns or undisturbed cores, respectively). An 

3 3 

average value of 2.60 g/cm (range from 2.52 to 2.69 g/cm ) was used for 

particle density. These values of particle density are typical for 
mineral soils (Brady 1974). The pore volume of each core was then cal- 
culated as shown below: 

««.«« w«iMm« f.r,T\ - pore space (%) „ total volume (ml) 
pore volume (ml) = ^^ ^ ^ x ^^ ^^^^ 

(4-2) 

An aliquot of each leachate sample was diluted (i.e., if neces- 
sary) in PBS containing 2% FCS and assayed directly for viruses by the 
plaque technique. In order to detect small numbers of viruses, the 
leachates from laboratory-packed soil columns which had received 
chemical sludges and lime-stabilized, ferric chloride sludge were 
concentrated by membrane filtration (Farrah et^ al_. 1976; Hill et a1 . 
1971; Shuval and Katzenelson 1972; Sobsey et al. 1973; Sobsey et al. 
1980b). These soil leachates were collected in 1/2 pore volume frac- 
tions which were assayed individually for viral infectivity as 
described above. Pore volumes 0.5 through 5.0 (and 5.5 through the 



106 



final pore volume) were subsequently combined and concentrated 160-fold 
as follows. The leachate sample was adjusted to pH 3.5 by the addition 
of 1 M glycine buffer, pH 2.0, and adjusted to a final concentration 
of 0.0005 M aluminum chloride. The treated sample was then passed 
through a series of 3.0- and 0.45-ym Filterite filters in a 47-mm 
holder. Adsorbed viruses were eluted from the filters with 7 ml of PBS 
containing 10% FCS, pH 9.0. The filter eluate was adjusted to neutral 
pH by the addition of 1 M glycine buffer, pH 2.0, and assayed for 
viruses by the plaque technique. 

The quantity of poliovirus detected in each soil leachate was 
expressed as a percentage of the amount of virus applied to the soil. 
For cores receiving poliovirus continuously (i.e., laboratory-packed 
soil columns), the quantity of poliovirus leached was expressed as 
the cumulative percent of the total viral PFU having been applied at 
each pore volume and it was calculated by the following equation: 



poliovirus eluted at pore 
volume b (as cumulative % 
of total PFU having been 
applied at pore volume b) 



pore volume (ml) 



pv=b 



poliovirus eluted in 



r [ju Muv I rub ei uuea in 
pv=l/a ^^^^ ^^^ P°'^^ volume (PFU/ml) 



I 



b X pore volume (ml ) 



influent poliovirus 
concentration (PFU/ml) 



X 100 



(4-3) 



where pv represents the pore volume number; the leachate samples were 
collected and assayed in 1/a pore volume fractions (a set at 1, 2, or 3 



107 



in the research reported herein); and b can be set at any value between 
1/a and the final pore volume number collected. For cores receiving 
poliovirus in a spiked fashion (i.e.. all at once at the beginning of 
the experimental trial; used this procedure in both laboratory-packed 
soil columns and undisturbed soil cores), the quantity of poliovirus 
leached was expressed as the cumulative percent of the total viral PFU 
applied and it was calculated by the following equation: 



poliovirus eluted at pore 
volume b (as cumulative % 
of total PFU applied) 



pore volume (ml) 



y poliovirus eluted in 
pv=l/a ^^^^ ^/^ pore volume (PFU/ml) 



poliovirus applied (total PFU) 



X 100 



(4-4) 



where pv, a, and b are defined as in Equation (4-3) above. 

The pH and conductivity of each soil core leachate were measured 
as described above for undiluted digested sludges. 

Distribution of virus in the soil . The distribution of polio- 
virus in the soil profile was studied in two 27-cm laboratory-packed 
columns of Eustis fine sand. These soil columns were prepared and 
treated with virus-seeded, diluted (1:50 with 0.01 N calcium chlor- 
ide or dtstilledvvatec) anaerobically digested sludge as described 
above. The leachates from these columns were collected and assayed for 
viruses as described earlier. Following leaching, the soil profile in 



108 



each column was separated into 2- to 3-cni sections. The sludge solids 
resting on top of the soil were also separated and considered as one 
section. Each soil (or sludge solids) section was well mixed in a 
sterile beaker with a spatula, wet weighed, and then a representative 
sample (10 grams of wet soil; total known amount of top wet sludge 
solids) was taken and subjected to the following virus recovery method- 
ology. Each sample was mixed with 3% (wt./vol.) beef extract (Difco 
Laboratories, Detroit, Michigan), buffered with Tris(hydroxymethyl)» 
aminomethane (Sigma Chemical Co., St. Louis, Missouri) at pH 9.0 in the 
proportion of 1 gram of wet soil (or wet sludge solids) per 2 ml of 
eluent. This solution has been found to be effective in the elution of 
poliovirus type 1 from soil (Bitton et a]_. 1979a) and from sludge solids 
(Farrah et aj^. -igSlb). The mixtures were then vortexed for 30 seconds 
and sonicated for 3 minutes at maximum deflection (60 watts) using a 
Branson sonifijer (Branson Instruments Inc., Danbury, Connecticut). The 
samples were then centrifuged at 1900 x g for 10 minutes at 4°C. The 
supernatants were adjusted to neutral pH. An aliquot of each super- 
natant produced was then diluted (i.e., if necessary) in PBS containing 
2% PCS and assayed directly for viruses by the plaque technique. The 
amount of poliovirus recovered was expressed as PFU per soil (or sludge 
solids) section. 

Effect of Soil Bulk Density on Poliovirus Transport 

The poliovirus transport studies described above were conducted 
with laboratory-packed soil columns and undisturbed soil cores displaying 
similar bulk densities as found in the field for the two soils studied 



109 



(see Table 4-3). In the laboratory-packed columns, the field bulk 
densities were accurately reproduced as detailed earlier. The undis- 
turbed soil cores were obtained in a manner that preserved the natural 
structure and bulk density of the soil (see page 102 above). In fact, 
undisturbed soil cores are frequently taken in order to measure the 
field bulk density of a soil (Blake 1965). However, the soil in 
undisturbed cores can be compressed during sampling and thereby result 
in an increase in bulk density as compared to the field bulk density 
(Blake 1965; Funderburg et al_. 1979). For example, compression of the 
soil is likely to occur when the soil is wet during sampling (Blake 
1965). Although the undisturbed cores used in this study were carefully 
sampled so as to not compress the soil, it was deemed important to 
determine what effect, if any, compression of the soil (i.e., increase 
in bulk density) might have on the transport of poliovirus. Laboratory- 
packed soil columns of Red Bay sandy loam subsoil (consisted mainly of 
the A2 and Bit horizons--see Table 4-3) were used in these experiments 
and they were prepared at different bulk densities as described below. 

Soil moisture content-bulk density curve . If the compactive force 
is held constant, the density to which a given soil can be compacted 
increases with a corresponding increase in the soil moisture content 
up to the optimum moisture level (Felt 1965). Increases in soil 
moisture content beyond this level result in reductions in the soil 
bulk densities achieved (Felt 1965). A soil moisture content-bulk 
density curve was produced for the Red Bay sandy subsoil using the pro- 
cedure of Wilson (1950). Briefly, the procedure consisted of adjusting 



no 



air-dried soil to moisture contents ranging from 7.3% to 14.4% (wt./wt.) 
using rain water and then compacting each sample in the Harvard compac- 
tion apparatus (Soil test Inc., Evanston, Illinois). Each soil sample 
was added to the compaction mold (known volume) in 3 layers with a 
compactive force of 10 tamps applied per layer (at different positions) 
using a 20-1 b (9.1 -kg) tamper (Soil test Inc., Evanston, Illinois; see 
Wilson 1950). The soil sample was then ejected from the mold, wet 
weighed, and dried to constant weight. The exact soil moisture content 
and dry compacted bulk density were calculated for each sample. The 
soil moisture content-bulk density curve obtained for the Red Bay sandy 
loam subsoil is shown in Figure 4-4. For the compactive force used, a 
maximum bulk density of 1.96 g/cm was achieved when the soil moisture 
content was 12.4% (i.e., optimum moisture content). It should be noted 
that cohesionless soils (i.e., sands) are compacted to maximum density 
by simply vibrating the air-dried soil (Felt 1965). However, the 
increase in bulk density achieved by maximum compaction is much smaller 
for sands than for finer textured soils (Brady 1974; Freeze and Cherry 
1979). This phenomenon was observed with the Eustis fine sand subsoil 
(consisted mainly of the A21 and A22 horizons— see Table 4-3) for which 
the maximum bulk density attained was only 1.70 g/cm"^. Thus, the Red 
Bay sandy loam subsoil was used here because of the greater range in bulk 
densities which could be produced with this soil. 

Saturated hydraulic conductivity . Prior to initiating polio- 
virus transport studies, it was important to determine the effect com- 
paction (i.e., increase in bulk density) of the Red Bay sandy loam 



m 



2.0 



oo 



E 



4-> 
•r- 
M 

e 
■o 



1.9 



1.8 - 



1 1 1 1 r 



o 

C/0 



1.7 



1.6 




Maximum bulk density = 1.96 g/cm" 

Optimum moisture con- 
tent for compaction = 12.4% 



1 



I 



± 



± 



8 9 10 11 12 13 
Soil moisture content (%) 



14 



15 



FIGURE 4-4. Soil moisture content-bulk density curve for the Red 
Bay sandy loam subsoil 

Air-dried samples of Red Bay sandy loam subsoil 
(i.e., consisting of the A2 and Bit horizons—see 
Table 4-3) were adjusted to moisture contents 
ranging from 7.3% to 14.4% (wt/vit.). The soil 
samples were then compacted using the procedure 
of Wilson (1950). 



112 



subsoil would have on the rate of water movement through the soil. 
The soil was packed into acrylic plastic columns (10 cm in length and 
4^cm internal diameter; packed 5.0 cm with soil) at bulk densities 
of 1.45, 1.60, 1.70, 1.85, and 2.00 dry g/cm . A polypropylene screen 

(105-ym pore size) was used to support the soil in each column. The 

3 
columns at bulk densities of 1.45 and 1.60 g/cm were packed with 

air-dried soil while tapping on the outside of the columns. The amount 

of tapping was increased to achieve the higher bulk density (i.e., 

3 
1.60 g/cm ). The columns at bulk densities of 1.70, 1.85, and 2.00 

3 
g/cm were packed with moist soil [12.4% (wt./wt.) moisure content was 

adjusted with rain water]. As shown above, 12.4% is the optimum 

moisture content for the compaction of this soil. The moist soil 

was compacted in the soil columns in 3 layers with 13, 20, or 33 tamps 

applied per layer [using a 20-lb (9.1-kg) tamper as described by 

Wilson (1950)— see above] in order to obtain a bulk density of 1.70, 

3 
1.85, or 2.00 g/cm , respectively. The saturated hydraulic conduc- 
tivity of each soil column was then measured using the "constant-head" 
method of Klute (1965). Briefly, the procedure consisted of applying 
a constant, hydraulic head of 2.5 cm to each saturated soil column using 
rain water and then measuring the volume of leachate collected in a 
measured time. When possible, the leachate sample was collected 
within 30 minutes of the beginning of leaching as recommended by Klute 
(1965). The saturated hydraulic conductivity was calculated by the 
following equation (Klute 1965): 

K = (Q/At)(L/AH) (4-5) 



113 



where 

K = saturated hydraulic conductivity (in cm/hr) 

Q = volume of leachate (in cm ) 

A = cross-sectional area of soil sample (18.09 cm for 

the soil columns in this study) 

t = time to collect volume of leachate (in hr) 

L = length of soil sample (5.0 cm) 

AH = hydraulic head difference (7.5 cm) 

From Table 4-4, it is clear that the saturated hydraulic conductivity 
of the Red Bay sandy loam subsoil decreased as the bulk density of the 
soil increased from 1.45 to 2.00 g/cm . At bulk densities of 1.70 g/cm 
or greater, the saturated hydraulic conductivities were drastically 
reduced (see Table 4-4). Therefore, soil columns packed a these high 
bulk densities (i.e.,>1.70 g/cm ) could not be used in subsequent 
poliovirus transport experiments because of the large amount of time 
required to collect an adequate volume of leachate. 

Poliovirus transport . The effect of soil bulk density on 
poliovirus transport was studied using laboratory-packed soil columns 
of Red Bay sandy loam subsoil. The air-dried soil (not autoclaved) 
was packed into acrylic plastic columns (10 cm in length and 4.8-cm 

internal diameter; packed 10 cm with soil) at bulk densities of 1.45 

3 
and 1.60 g/cm while tapping on the outside of the columns. The charac- 
teristics of the soil columns packed at these bulk densities are shown 

in Table 4-5. As the bulk density of the soil was increased from 1.45 

3 
to 1.60 g/cm , there was a corresponding decrease in the percent pore 



114 



TABLE 4-4. 


Effect of soil bulk 
conductivity of the 


dens 
Red 


;ity on the saturated hydraulic 
Bay sandy loam subsoil 


Bulk 

density ^ 

(dry g/cm ) 


Saturated hydraulic 


conductivity 


Permeability 
class^ 




ml /mi n 




cm/hr^ 


1.45 




33 




73 


Very rapid 


1.60 




a. 2 




18 


Rapid 


1.70 




0.35 




0.77 


Moderately slow 


1.85 




0^ 







^ery slow 


2.00 




0^ 







Very slow 



The sample of Red Bay sandy loam subsoil used consisted mainly of 
the A2 and Bit horizons (see Table 4-3). The bulk densities shown were 
produced in soil columns (i.e., 10 cm in length and 4.8-cm internal 
diameter; packed 5.0 cm with soil) as described in the Materials and 
Methods section. 

The saturated hydraulic conductivity of each soil column was 
measured using the "constant-head" method of Klute (1965). 

Calculated using Equation (4-5). 

^According to Klute (1965). 

^No leachate passed through these columns in a 2-hour period. 



115 



TABLE 4-5. Characteristics of the 10-cm columns of Red Bay sandy 
loaniosubsoil packed at bulk densities of 1.45 and 1.60 
g/cm 



Bulk density^ Particle density Pore space^ Pore volume 
(g/cm-^) (g/cm^) (%) (ml) 



1.45 2.60 44.2 79.9 

1.60 2.60 38.5 69.6 



The sample of Red Bay sandy loam subsoil used consisted mainly of 
the A2 and Bit horizons (see Table 4-3). The bulk densities shown were 
produced in the soil columns (i.e., 10 cm in length and 4.8-cm internal 
diameter) by packing the appropriate grams of air-dried soil in the 
column volume. 

This particle density value is typical for mineral soils (Brady 
1974). 

^Calculated using Equation (4-1). 

Calculated using Equation (4-2). 



116 



space and pore volume of the lO-cm soil column (see Table 4-5). 
Three columns were prepared at each bulk density. A polypropylene 
screen (105-ym pore size) was used to support the soil in each column. 
The soil columns were placed in soil column holders and treated with 
carbon dioxide as described earlier (see page 97), The soil columns 
were then conditioned by passing 2 pore volumes of nonseeded primary 
wastewater effluent using the peristaltic pump. Following conditioning, 
poliovirus was suspended in primary wastewater effluent (as described 
on page 91) and subsequently applied continuously to the soil columns 
at approximately 3.5 ml/min using the peristaltic pump. As detailed 
earlier (see page 91 ), influent poliovirus concentration was determined 
from viral assays made at the beginning and end of each column experiment. 
After percolation through the soil, the column leachates were collected 
in pore volumes using sterile screw-capped bottles. The poliovirus 
content, pH and conductivity of the leachates were determined as 
described earlier (see pages 104-107) . 

Statistical treatment of data . Statistical treatment of the 
data was performed with the use of a Hewlett-Packard calculator model 
9810A and Statistics Package V-6 (Hewlett-Packard Company, Loveland, 
Colorado). 

Results and Discussion 
Prior to conducting field experiments, it appeared necessary 
to study the transport pattern (i.e., movement or retention) of sludge- 
associated viruses in the soils under consideration, Eustis fine sand 
and Red Bay sandy loam. Experiments were undertaken to study viral 



117 



transport under optimal conditions (i.e., in the presence of 0.01 N 
CaCl2) and under more realistic conditions involving sludge applica- 
tion to soils. 

Poliovirus Suspended in 0.01 N C aCl2 

Red Bay sandy loam . The "retention potential" of this soil 
towards poliovirus was first determined, under optimal conditions, in 
the presence of 0.01 N CaCl2. As shown in Table 4-6, more than 99.99% 
of the viral load was removed, presumably due to adsorption, after 
10 pore volumes of solution had passed through the soil. The pH of the 
soil solution varied from 5.1 to 6.3 and the conductivity was around 
1300 pmhos/cm (see Table 4-6). A shift from calcium chloride to rain 
water did not result in any appreciable release of soil-bound viruses 
although the conductivity of the soil solution decreased from 1320 to 
54 ymhos/cm. It is well known that divalent cations, at appropriate 
concentrations, enhance the adsorption of viruses to soils (Bitton 
1975; Drewry and Eliassen 1968; Gerba et a^. 1975; Lefler and Kott 
1974). Rain water may be important in the redistribution and transport 
of viruses through the soil matrix. This has been suggested in the 
field (Wei lings et al. 1975) and demonstrated in the laboratory 
(Duboise et al. 1976; Lance et al_. 1976). However, it was found that 
rain water did not significantly affect the desorption of viruses from 
a soil containing 28% clay (Scheuerman et al. 1979). 

Eustis fine sand . The "retention potential" of this soil 
towards poliovirus was also evaluated, as described above for the Red 
Bay sandy loam, in the presence of 0.01 N CaClp. It was observed 



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(see Table 4-7) that the maximum breakthrough per pore volume was 0.3%. 
This table also shows that approximately 99.3% of the viral load was 
retained by this soil following leaching with 10 pore volumes of 
seeded 0.01 N CaCl2- As in the case of the Red Bay sandy loam (see 
Table 4-6), this sandy soil has displayed a substantial capability of 
removing poliovirus suspended in a calcium chloride solution. 

Poliovirus Suspended in Diluted An- 
aerobically Digested Sludge 

Red Bay sandy loam . Anaerobically digested sludge (2% solids 
content, w/v), diluted 1 to 50 (v/v) with 0.01 N calcium chloride or 
distilled water, was seeded with poliovirus and thoroughly mixed to 
bring about the adsorption of the virus to the sludge particles. The 
virus-sludge mixtures were then pumped onto the top of Red Bay sandy 
loam columns. The dilution of the sludge (1:50) with calcium chloride 
or distilled water was necessary in order to facilitate its delivery 
by the pump. Under these conditions, 10 pore volumnes of the diluted 
sludge corresponded to the application of 2.5 cm (1 inch) of anaerob- 
ically digested sludge containing 2% solids (w/v). Table 4-8 describes 
the movement of poliovirus suspended in sludge diluted in 0.01 N calcium 
chloride. No virus breakthrough was detected prior to the seventh pore 
volume. At the tenth pore volume, only 0.01% of the total virus applied 
appeared in the soil effluent. The subsequent addition of two pore 
volumes of sterile rain water (which is the equivalent of 25 cm of 
rain) did not elute adsorbed viruses (see Table 4-8). In fact, at the 
12th pore volume, the percent of total viruses applied which appeared 
in the leachate remained at 0.01. Table 4-9 displays the results 



121 



TABLE 4-7. Retention of poliovirus type 1 by a packed column of 
Eustis fine sand subsoil when suspended in 0.01 N 
CaClo 



No. of pore 

volumes^ 

eluted 



1 
2 
3 
4 
5 
6 
7 
8 
9 

10 

11 



Poliovirus 

eluted 

(PFU/ml) 



% of Influent^ 

poliovirus 
concentration 






4.2 


4.2 




25 
21 










0.06 



0.06 





0.3 

0.3 







One pore volume for the column used equals 71 ml. The labora- 
tory-packed column was 10 cm in length and 4.8 cm internal diameter. 
The sample of Eustis fine sand subsoil used consisted mainly of the 
A21 and A22 horizons (see Table 4-3). The column was conditioned 
with 5 pore volumes of 0.01 N CaCl2. The solution was applied con- 
tinuously to the column at approximately 5 ml/min using a peristaltic 
pump (Buchler, Fort Lee, N.J.) . 

Poliovirus was seeded in the influent (i.e., 0.01 N CaClo) at 
at concentration of 7.3 x 10^ PFU/ml. The conductivity of O.of 
N CaCl2 was 1210 pmho/cm at 25°C and the pH was 6.4. 



122 



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126 



pertaining to the movement of poliovirus through the Red Bay sandy 
loam treated with sludge which had been diluted in distilled water. 
No virus breakthrough was detected in the soil leachates following 
percolation of 8,5 pore volumes. The application of sludge diluted 
with distilled water resulted in a gradual soil clogging and the per- 
colation experiment was ended when only 8.5 pore volumes had been 
collected. In this particular experiment, the specific conductance was 
around 90 ymhos/cm and was probably sufficient to promote virus adsorp- 
tion to the soil . 

Eustis fine sand . The transport pattern of poliovirus suspended 
in anaerobically digested sludge diluted in distilled water or 0.01 N 
calcium chloride was next evaluated using columns of Eustis fine sand. 
As shown in Table 4-10, poliovirus suspended in sludge diluted in 
distilled water was found to rapidly move thorugh the soil and appear 
in the column effluent. Breakthrough occurred by the first pore 
volume (0,1%) and reached a maximum at the seventh pore volume of 39.9% 
of the influent poliovirus concentration. Fractions beyond the seventh 
pore volume could not be collected because of clogging of the column. 
The results for sludge diluted with 0.01 N calcium chloride, on the 
other hand, show no virus breakthrough by the ninth pore volume beyond 
which the column became clogged (see Table 4-10). Thus, it was observed 
that the change in the ionic composition of sludge when diluted in 
distilled water allowed for rapid virus transport through the soil 
column. It is postulated that a reduction in the specific conductance 
of the sludge diluted in distilled water resulted in poor virus 



127 



TABLE 4-10. Movement or retention of poliovirus type 1 when suspended in 
anaerobically digested sludge diluted (1:50) with distilled 
water or 0.01 N CaClp. respectively, and applied to 10 cm 
packed columns of Eustis fine sand subsoil 



No. of pore 

volumes^ 

eluted 



Poliovirus 

eluted 

(PFU/ml) 



% of Influent^ 

pol iovirus 
concentration 



Conductivity 

of pore 
volume collected 
(ymho/cm at 25°C) 



pH of pore 

volume 
collected 



Column l--applied sludge^ diluted (1:50) with distilled water 



1 


5.0 


X 


10' 


0.1 


2 


3.3 


X 


10^ 


0.1 


3 


1.4 


X 


10^ 


2.4 


4 


7.4 


X 


10^ 


12.5 


5 


1.7 


X 


104 


28.2 


6 


2.1 


X 


10^ 


36.2 


7 


2.4 


X 


10^ 


39.9 



46 


7.0 


102 


6.8 


250 


6.6 


280 


5.9 


300 


5.8 


310 


5.8 


310 


5.5 



Column 2--applied sludge^ diluted (1:50) with 0.01 N CaCl2 



1 
2 
3 

4 
5 
6 
7 























1230 


5.1 


1290 


5.2 


1310 


5.2 


1320 


5.2 


1360 


5.3 


1300 


5.3 


1250 


5.4 



TABLE 4rl0. Continued. 



128 



No. of pore 

vol ume $3 

eluted 


Poliovirus 

eluted 

(PFU/ml) 


% of Influent^ 

poliovirus 
concentration 


Conductivity 
of pore 
volume collected 
(pmho/cm at 25°C) 


pH of pore 

vol ume 
collected 


8 
9 










1300 
1400 


5.5 
5.4 



^One pore volume for the columns used equals 71 ml. The laboratory- 
packed columns were 10 cm in length and 4.8 cm internal diameter. The 
sample of Eustis fine sand subsoil used consisted mainly of the A21 and 
A22 horizons (see Table 4-3). Columns 1 and 2 were conditioned with 
5 pore volumes of distilled water and 0.01 N CaCl2, respectively. All 
solutions were applied continuously to the columns at approximately 
5 ml/min using a peristaltic pump (Buchler, Fort Lee, N.J.). 

Poliovirus was seeded in the influents of column 1 (i.e., sludge 
diluted with distilled water) and column 2 (i.e., sludge diluted^with 
0.01 N CaCl2) at concentrations of 5.9 x 104 PFU/ml and 2.6 x 10^ 
PFU/ml, respectively. 

^The anaerobically digested sludge (PDAN--see Table 3-2) used had 
a solids content of 1.4% and a pH of 7.2. Chemical parameters were not 
measured for the sludge diluted (1:50) with distilled water or 0.01 N CaCl2. 



129 



adsorption to the sludge and soil particles. Poliovirus retained by a 
Eustis fine sand column treated with sludge diluted with 0.01 N calcium 
chloride was not eluted with rain water (see Table 4-11). It appears, 
therefore, that adsorbed viruses in sludge- treated soils are not readily 
displaced and transported further down the soil profile by a solution 
low in ionic strength such as rain water. 

The distribution of poliovirus within Eustis fine sand columns 
was investigated next. For this purpose, 29-cm columns were packed 
with 27 cm of this soil, and poliovirus was subsequently applied to the 
soil surface while suspended in sludge diluted (1:50, v/v) in distilled 
water or 0.01 N CaCl2. Table 4-12 shows the transport patterns of 
poliovirus in 27-cm soil columns that had been treated with the diluted 
sludges. Similar trends were previously observed with the 10-cm 
columns (see Table 4-10). No virus breakthrough was observed in the 
column treated with sludge diluted with 0.01 N CaClpj whereas the 
column receiving sludge diluted with distilled water was found to 
display virus in the leachate after the third pore volume (see Table 
4-12). The experiment was stopped by the fourth pore volume because 
the column receiving the sludge diluted with distilled water became 
clogged. The columns were subsequently sectioned (i.e., after allowing 
ponded water to soak in overnight) to study virus distribution within 
the soil (see Tables 4-13 and 4-14). It can be seen that in the 
column treated with sludge diluted with 0.01 N calcium chloride, polio- 
virus was found in the packed sludge and in the top 5 cm of soil (see 
(Table 4-13). The results for the column receiving sludge diluted with 



130 



TABLE 4-11. Retention of poliovirus type 1 by a 10 cm packed column of 
Eustis fine sand subsoil when suspended in anaerobically 
digested sludge diluted (1:50) with 0.01 N CaCl2, and after 
subsequent application of rain water and 0.01 N CaCl2 



No. of pore 

volumes^ 

eluted 


Poliovirus 

eluted 

(PFU/ml) 


% of Influent*^ Conductivity 

poliovirus of pore 
concentration volume collected 
(ymho/cm at 25°C) 


pH of pore 

volume 
collected 


Sludge*^ diluted (1:50) with 0.01 


N CaCl2 and seeded with 


poliovirus 


1 







1280 




4.7 


2 







1335 




4.9 


3 







1 380 




4.9 


4 







1400 




5.0 


5 







1430 




5.1 


6 







1430 




5.2 


7 







1430 




5.3 


8 







1420 




5.2 


9 







1420 




5.2 


Shift to nonseeded rain 


water 








10 







1360 




5.1 


n 







315 




5.0 


12 







150 




5.0 


13 







88 




6.6 


14 







63 




6.6 



131 



TABLE 4-11. Continued, 



No. of pore 

volumes^ 

eluted 



Poliovirus 

eluted 

(PFU/ml) 



% of Influent^ 

pol iovirus 
concentration 



Conductivity 
of pore 
volume collected 
(ymho/cm at 25°C) 



pH of pore 

volume 
collected 



Shift to nonseeded 0.01 N CaCl^^ 



15 








16 








17 


22 


0.1 


18 








19 









60 

1010 
1240 
1320 
1320 



6.5 
5.2 
5.2 
5.2 
5.1 



Shift to nonseeded rain water 



20 








21 








22 








23 








24 









1200 


5.1 


350 


5.9 


88 


6.5 


64 


6.8 


58 


6.8 



One pore volume for the column used equals 71 ml. The laboratory- 
packed column was 10 cm in length and 4.8 cm internal diameter. The 
sample of Eustis fine sand subsoil used consisted mainly of the A21 and A22 
horizons (see Table 4-3). The column was conditioned with 5 pore volumes 
of 0.01 N CaClp. All solutions were applied continuously to the column at 
approximately 5 ml/min using a peristaltic pump (Buchler, Fort Lee, N.J.). 

Poliovirus was seeded in the influent (i.e., sludge diluted with 
0.01 N CaCl2) at a concentration of 2.6 x 10"+ PFU/ml. 

The anaerobically digested sludge (PDAN— see Table 3-2) used had a 
solids content of 1.4% and a pH of 7.2. Chemical parameters were not 
measured for the sludge diluted (1:50) with 0.01 N CaCl2. 

Rain water was collected next to the Environmental Engineering 
Sciences building at the University of Florida, Gainesville. See Table 
4-2 for chemical characteristics of the rain water. 



The conductivity and pH of 0.01 N CaCl2 was 1210 ymho/cm at 25°C 
and 6.4, respectively. 



132 

TABLE 4-12. Movement or retention of poliovirus type 1 when suspended in 
anaerobically digested sludge diluted (1:50) with distilled 
water or 0.01 N CaCl2. respectively, and applied to 27 cm 
packed columns of Eustis fine sand subsoil 



No. of pore 

volumes^ 

eluted 


Poliovirus 

eluted 

(PFU/ml) 


% of Influent"^ 

poliovirus 
concentration 




Conductivi 
of pore 
volume coll 
(ymho/cm at 


ty 

ected 
25°C) 


pH of pore 

volume 
collected 


Column 


1- 


-appl 


ied 


si 


udge^ 


diluted (1 


:50) 


wi 


th distilled water 




0.5 




















29 






6.6 


1.0 




















29 






6.3 


1.5 




















51 






6.2 


2.0 




















61 






6.2 


2.5 




















104 






6.5 


3.0 




















77 






6.5 


3.5 






2.5 


X 


102 


1.8 






78 






5.6 


4.0 






2.7 


X 


10^ 


1.9 






— 






— 


Column 


2- 


-appl 


ied 


si 


udge^ 


diluted (1 


:50) 


wi 


th 0.01 N CaCl2 






0.5 




















1180 






4.8 


1.0 




















1270 






4.8 


1.5 




















1270 






4.7 


2.0 




















1300 






4.8 


2.5 




















1260 






4.9 


3.0 




















1300 






4.9 



133 



TABLE 4-12. Continued. 



No. of pore 

volumes^ 

eluted 


Poliovirus 

eluted 

(PFU/ml) 


% of Influent*^ 

poliovirus 
concentration 


Conductivity 
of pore 
volume collected 
(pmho/cm at 25°C) 


pH of pore 

volume 
collected 


3.5 
4.0 










1310 
1310 


5.0 
5.0 



One pore volume for the columns used equals 192 ml. The laboratory- 
packed columns were 29 cm in length and 4.8 cm internal diameter; the 
columns were filled only 27 cm with soil (2 cm left on top for packed 
sludge). The sample of Eustis fine sand subsoil used consisted mainly of 
the A21 and A22 horizons (see Table 4-3). Columns 1 and 2 were condi- 
tioned with 5 pore volumes of distilled water and 0.01 N CaClo, respective- 
ly. All solutions were applied continuously to the columns at approxi- 
mately 5 ml/min using a peristaltic pump (Buchler, Fort Lee, N.J.). 
Following elution, the columns were sectioned as seen in Tables 4-13 and 
4-14. 



Poliovirus was seeded in the influents of column 1 (i.e 



diluted with distilled water) and column 2 (i.e., sludge diluted with 
0.01 N CaCl2) at concentrations of 1.4 x 10^ PFU/ml and 1 
respectively. 



sludge 

V 

7 X 10^ PFU/ml, 



^The anaerobically digested sludge (PDAN--see Table 3-2) used had a 
solids content of 1.4% and a pH of 7.2. Chemical parameters were not 
measured for the sludge diluted (1:50) with distilled water or 0.01 N CaCl2. 



134 



TABLE 4-13. Distribution of poliovirus type 1 in the soil profile 
of a 27-cni packed column of Eustis fine sand which had 
received virus-seeded, anaerobically digested sludge 
diluted (1:50) with 0.01 N CaCU 



Depth in 






Poliovirus recovered from 


soil^ 


CO 1 umn vcm; 


PFU/g of 
wet soil 


PFU/soil 
section 


% of Total 
PFU applied^ 


Top Sludge 


38 


,200 


2.1 X 10^ 


1.6 


0-3 


1 


,000 


9.9 X 10^ 


0.8 


3-5 




80 


8.1 X 10^ 


0.6 


5-7 













7-9 













9-11 













11-13 













13-15 













15-17 













17-19 













19-21 













21-23 













23-25 













25-27 














^The labora-tory-packed column.was 29 cm in length and 4.8-cm 
internal diameter; the column was filled only 27 cm with soil (2 cm 
left on top for the packed sludge solids). The column was treated 
with virus-seeded diluted sludge as described in Table 4-12. The 
soil column was then sectioned and virus was eluted from each soil 
section. 



135 
TABLE 4-13. Continued. 



Each soil section was mixed well, and a 10-gram wet sample was 
taken and mixed with 20 ml of 3% beef extract, Tris buffered at pH 9.0. 
This mixture was then vortexed for 30 sec and sonicated for 3 min. 
The sample was then centrifuged at 1900 x g for 10 min at 4°C. The 
supernatant was subsequently assayed for viruses. 

r 7 

The amount of virus applied to the soil column was 1.3 x 10 

total PFU (see Table 4-12). Overall recovery of the virus applied was 
3.0% [i.e., 3.0% found in the soil and 0% found in the soil leachates 
(see Table 4-12)]. 

Refers to the sludge solids resting on top of the soil. The 
total amount of these solids was separated, subjected to the same virus 
elution method as the soil (added eluent at the proportion of 2 ml per 
gram of wet sludge solids) and was considered as one section. 



136 



TABLE 4-14. Distribution of poliovirus type 1 in the soil profile of 

a 27-cm packed column of Eustis fine sand which had 

received virus-seeded, anaerobically digested sludge 
diluted (1:50) with distilled water 



Depth in 
column^ (cm) 




Poliovirus recovered from 


soil^ 


PFU/g of 
wet soil 


PFU/soil 
section 


% of Total 
PFU applied'^ 


Top Sludge 


4,434 


4.9 X 


10^ 


0.4 


0-3 


2.720 


2.9 X 


10^ 


2.6 


• 3-5 


2,940 


2.5 X 


10^ 


2.3 


5-7 


2,266 


2.2 X 


10^ 


2.0 


7-9 


966 


7.3 X 


10^ 


0.7 


9-11 


1,694 


1.3 X 


10^ 


1.2 


11-13 


1,106 


9.8 X 


10^ 


0.9 


13-15 


734 


6.4 X 


10^ 


0.6 


15-17 


874 


6.8 X 


10^ 


0.6 


17-19 


260 


2.2 X 


10^ 


0.2 


19-21 


614 


4.0 X 


10^ 


0.4 


21-23 


534 


5.2 X 


10^ 


0.5 


23-25 


46 


3.4 X 


10^ 


0.03 


25-27 


60 


5.8 X 


10^ 


0.05 . 



^The laboratory-packed column was 29 cm in length and 4.8-cm 
internal diameter; the column was filled only 27 cm with soil (2 cm 
left on top for the packed sludge solids). The column was treated 
with virus-seeded diluted sludge as described in Table 4-12. The 
soil column was then sectioned and virus was eluted from each soil 
section. 



137 



TABLE 4-14. Continued 



Each soil section was mixed well, and a 10-gram wet sample was 
taken and mixed with 20 ml of 3% beef extract, Tris buffered at pH 9.0. 
This mixture was then vortexed for 30 sec and sonicated for 3 min. 
The sample was then centrifuged at 1900 x g for 10 min at 4°C. The 
supernatant was subsequently assayed for viruses. 

r 7 

The amount of virus applied to the soil column was 1.1 x 10 

total PFU (see Table 4-12). Overall recovery of the virus applied was 

13.0% [i.e., 12.5% found in the soil and 0.5% found in the soil leachates 

(see Table 4-12)]. 

Refers tothesludge solids resting on top of the soil. The total 
amount of these solids was separated, subjected to the same virus 
elution method as the soil (added eluent at the proportion of 2 ml per 
gram of wet sludge solids) and was considered as one section. 



138 



distilled water, on the other hand, show that poliovirus was distributed 
throughout the length of the column with slightly higher concentrations 
in the top 3 cm of soil (2.6% of the total PFU applied--see Table 
4-14). Thus, the results presented indicate that, because of the 
appropriate ionic environment, poliovirus was adsorbed in the top of 
the column receiving sludge diluted with 0.01 N calcium chloride. How- 
ever, sludge diluted with distilled water affected the absorption 
process and allowed virus to move down the column, ultimately appearing 
in the effluent (see Table 4-12). 

Poliovirus Suspended in Sludge Liquor 

The centrifugation of anaerobically digested sludge resulted in 
a supernatant that will be referred to as sludge liquor. This liquor 
had a conductivity of 1580 ymhos/cm at 25°C and its pH was 8.1. Other 
characteristics of this liquor are displayed in Table 4-1. In order to 
stress the importance of sludge solids in virus movement through soils, 
soil column experiments were undertaken which involved viruses sus- 
pended in sludge liquor. 

Red Bay sandy loam . Poliovirus was suspended in sludge liquor 
and subsequently was applied continuously to a 10-cm column of Red Bay 
sandy loam subsoil. In Figure 4-5, the breakthrough curve obtained is 
shown and it is seen that 33.7% of the total virus applied had appeared 
in the leachate by the seventh pore volume. The conductivity of the 
leachates did not vary significantly from the conductivity of the 
sludge liquor (i.e., 1580 ymho/cm at 25°C). The pH of the leachates 
varied from approximately 5.0 to 6.3. Moreover, it is seen in Table 4-1 



FIGURE 4-5. Movement of poliovirus type 1 through a 10 cm packed 
column of Red Bay sandy loam subsoil when suspended 
in anaerobically digested sludge liquor 

One pore volume for the column used equals 80 ml. 
The laboratory-packed column was 10 cm in length 
and 4.8 cm internal diameter. The sample of Red 
Bay sandy loam subsoil used consisted mainly of 
the A2 and Bit horizons (see Table 4-3). The 
column was conditioned with 2 pore volumes of sludge 
liquor. Poliovirus was then suspended in the sludge 
liquor at a concentration of 1.9 x 10^ PFU/ml and 
applied to the column. All solutions were applied 
continuously to the column at approximately 5 ml/min 
using a peristaltic pump (Buchler, Fort Lee, N.J.). 
The sludge liquor was produced by centrifuging 
anaerobically digested sludge (GDAN— see Table 3-2; 
solids content, conductivity and pH equal to 
2.0%, 3250 umho/cm at 25°C and 8.3, respectively) 
at 14,000 X g for 10 min at 4°C. This procedure 
was performed again on the decanted supernatant 
and this yielded the clear sludge liquor. The 
conductivity of the sludge liquor was 1580 umbo/ 
cm at 25°C and the pH was 8.1 (see Table 4-1 
for other chemical parameters). 



140 



> 



36 



33 



30 



Its 
4-' 


27 


o 




+J-— s 




<u 




M- E 




O 3 






•<'4 


&« O 




> 




OJ 




> (D 




■r- S_ 
4-) O 


?1 


(0 Q. 




n™ 




3 -SZ 




E U 




3 (0 




O OJ 


18 


(/5 -t-> 




n3 (0 




■a T3 

CU OJ 


15 


(/I •!- 




to 1 — 




OJ Q. 




S- Q. 






12 


<u c 




^— a; 




<u 




-o XJ 




<u 




3 


9 


r— 




<U 




(/) 




3 


6 


•r™ 




> 




o 




•r- 




"o 


3 


Q. 





o 

o 

tn 
c\i 

> E 
•I- u 

+-) -^ 
u o 

3 ^ 

t: e 
o 
o 




20 



15 



10 



- ^— — -o Conductivity 



Cumulative % 
3 PFU/ml 




12 
-11 

-10 
9 

-J 8 



7 <^ 



6 -o 

O) 



5 " 

3 
S- 
•r— 

> 

4 .2 



o 

Q. 



2 3 4 5 6 

No. of pore volumes eluted 



3 
2 

1 



6,5 
6.0 
5.5 
5.0 



8 



141 



that the sludge liquor contained high levels of Na, K, Ca, Mg, and 
soluble salts which would be conducive to virus adsorption to soil. 
In spite of this favorable ionic environment, a dramatic virus break- 
through (33.7%) occurred. These data support the contention that the 
sludge liquor contained substances which strongly interfered with virus 
adsorption to this soil. Furthermore, in this experiment, sludge solids 
which can bind viruses in the top of the soil matrix were not added. 
In a similar experiment, poliovirus was suspended in sludge diluted 
with sludge liquor brought to a final calcium chloride concentration 
of 0.01 N and subsequently applied to the soil column. In Figure 4-6, 
the breakthrough of poliovirus is seen to have been reduced to 22.6% 
(from 33.7% with only sludge liquor) as a direct result of the presence 
of sludge solids. When the length of the soil column was increased 
to 27 cm from 10 cm, there was a further decrease in poliovirus break- 
through from 22.6% to 8.1% (see Figure 4-7). Dilution of sludge in its 
own liquor prevents extreme changes in the sludge properties. For 
example, the pH was unchanged (8.3) and the conductivity was only 
slightly reduced from 3250 to 2600 ymho/cm at 25°C. A direct compari- 
son can be made between the experiment in which 27-cm columns 
received sludge diluted with sludge liquor brought to a final CaCK 
concentration of 0.01 N (Figure 4-7) and the experiment in which simi- 
lar size columns were treated with sludge diluted in 0.01 N calcium 
chloride (Table 4-8). For these experiments, breakthroughs of polio- 
virus detected in the soil leachates at the tenth pore volume cor- 
responded to 8.1% and 0.01% of the total virus applied, respectively. 
Quite clearly, the sludge liquor contained substances which interfered 



V:-- 



FIGURE 4-6. Movement of poliovirus type 1 through a 10 cm packed 
column of Red Bay sandy loam subsoil when suspended 
in anaerobically digested sludge diluted (1:50) with 
sludge liquor containing 0.01 N CaCl2 

One pore volume for the column used equals 80 ml. 
The laboratory-packed column was 10 cm in length 
and 4.8 cm internal diameter. The sample of Red 
Bay sandy loam subsoil used consisted mainly of 
the A2 and Bit horizons (see Table 4-3). The 
column was conditioned with 2 pore volumes of 
sludge liquor containing 0.01 N CaCl2- Poliovirus 
was then suspended in the diluted sludge at a con- 
centration of 4.0 X 10^ PFU/ml and applied to the 
column. All solutions were applied continuously 
to the column at approximately 5 ml/min using a 
peristaltic pump (Buchler, Fort Lee, N.J.). The 
anaerobically digested sludge (GDAN— see Table 
3-2) used had a solids content of 2.0%, a con- 
ductivity of 3250 ymho/cm at 25°C and a pH of 8.3. 
The conductivity of sludge diluted (1:50) with 
sludge liquor containing 0.01 N CaCl2 was 2500 
ymho/cm at 25°C and the pH was 7.5. The sludge 
liquor was produced by centrifuging GDAN sludge 
at 14,000 X g for 10 min at 4°C. This procedure 
was performed again on the decanted supernatant 
and this yielded the clear sludge liquor (see 
Table 4-2 for chemical parameters). 



143 



24 



c 

> 
Ml 

a. 



O 



O E 



(U 


> 


> 




'1— 


OJ 


4J 


s. 


(O 


o 




Q. 


3 




E -E 


3 


O 


O 


to 




O) 


I/) 




(13 


+J 




(O 


-a 




(U 


■o 


10 


(U 


10 


•f— 


OJ 


^— 


S- 


CL 


Q. 


Q. 


X 


ta 


<u 






c 




(U 


-o 


a; 


<u 


J3 


■M 




3- 




r- 




« 




(A 




3 




S. 




•^ 




> 




o 




•r" 




f^ 




o 




o. 





o 

o 

Lf> 
CM 

4-> n3 

•r— 

> s 
•I- (J 
+J ^^ 
o o 

■O E 

O 
C_3CM 

O 



20 




25 



20 



15 



10 



_ O — 



Cumulative % 



— — O Conductivity 

-O- 




2 3 4 5 6 

No. of pore volumes eluted 



14.4 
13.2 

12.0 
10.8 
9.6 1 



_ 8.4cM 
^ o 



7.2 



T3 
CL) 

3 



6.0 <" 



lO 

3 
S- 



4.8 g 



o 
c 



3.6 
2.4 
1.2 




5.5 
_ 5.0 
- 4.5 

4.0 



8 



FIGURE 4-7. Movement of poliovirus type 1 through a 27 cm packed 
column of Red Bay sandy loam subsoil when suspended 
in anaerobically digested sludge diluted (1:50) with 
sludge liquor containing 0.01 N CaCl2 

One pore volume for the column used equals 225 ml. 
The laboratory-packed column was 29 cm in length 
and 4.8 cm internal diameter; the column was filled 
only 27 cm with soil (2 cm left on top for packed 
sludge). The sample of Red Bay sandy loam subsoil 
used consisted mainly of A2 and Bit horizons (see 
Table 4-3). The column was conditioned with 2 
pore volumes of sludge liquor containing 0.01 
N CaClo- Poliovirus was then suspended in the 
diluted sludge at a concentration of 7.0 x 10^ 
PFU/ml and applied to the column. All solutions 
were applied continuously to the column at approxi- 
mately 5 ml/min using a peristaltic pump (Buchler, 
Fort Lee, N.J.). The anaerobically digested sludge 
(GDAN— see Table 3-2) used had a solids content 
of 2.0%, a conductivity of 3250 ymho/cm at 25°C 
and a pH of 8.3. The conductivity of sludge 
diluted (1:50) with sludge liquor containing 0.01 
N CaClg was 2600 ymho/cm at 25°C and the pH was 
8.3. The sludge liquor was produced by centri- 
fuging GDAN sludge at 14,000 x g for 10 min at 4°C. 
This procedure was performed again on the decanted 
supernatant and this yielded the clear sludge 
liquor (see Table 4-2 for chemical parameters). 



145 




3 4 5 6 7 
No. of pore volumes eluted 





■o 




0) 


T3 


(/) >> • C +-> "O 


C 


C Cr— S- O'l- OOOl 


ro 


T3 O •.- I/IO) +->El 34-> 


(/) 


(U4-N 3r— C C -r-Dl/) 




.:.iS O •>- TTO 0-C0<— 0<UaJT3 3 


OJ 


(J 1. 0)0300(01 — U+->iO''-5 


C 


roajo>4-T3i— c:3cn3 •■- -a 


•r- 


Q.I— ^oc T-carocrs-tos-cio 


>♦- 


1 Q. 0) X +J 1 — 0) O 3 0) O) 




>)E<>J(/)CL C 4-O-M^^ 


10 


s-rocMcui/jLooQ-cnn: -r-r — +JQ- 


•r— 


ow)<:E3 •oEc:q.o>>t- I 


-U S- 


-M 3W1«* 3-1- <UI-i_T3T3 


(/) o 


(0 (U-o •— -a a. cn-o x i- c a; 


13 3 


i..c:c:oc4-aj 3c o-Eros- 


UJ o- 


Ol— r0><U0'>-04-raO 3. (U 


•r— 


^ ^1— T-T-OOJ S-4-> 


t- t— 


lO 1— cu+Jc:Q.+->i.>^o^tr)a;r— 


O 


r— 'CMS- 00.1— -tJ+J •>+->C\JT3-i- 


<u 


s-<CO(/)T-fa<oc-i-«^ •! — 4- 


c en 


dJOJ Q-rO+J 4->a)>i— EOO 


E -a 


-c-MO) 2(oaji/io->- o -CO) 


3 3 


1— (i)x:cM i.s--i- +J+JS_-a x: 


^~ r~- 


E+J (/J4-><Ui.>,0(OM-CE+-> 


O </) 


ro .C:3£=S<UJ33 rOE 


O 


..r-M-4JS-(i; Q. XJ-— +J M- 


c 


1 — ■ao-'-T-oto "OC>5CLf)r^o 


-o o 


E 2>cc(a<i;o.— lO"*-;!- 


<u o 


r— >, OOO OUOJ+J- >> 


^ 01 


>— (Or— ■a-i-cj'"-ai3 >roO(a+-> 


O ro 


I^CCCUi — ^->CT3 "T-C •!- 


it> 1— 


S--r-COfa3-f-0+->-MS-'4-C> 


Q. 


tOOJrOOQ. r— i/)S_CU<l)0'i--i- 


T3 


1— -UE'r- 4->03Q.(UajQ. +J 


E <U 


roc 4->ajrOi/1 +JQ.3(/1 ^- (J 


U -tJ 


3'i--0-r-J= CCOCr/li/ld) "3 • 


i/> 


O" (UTSI— S-r-'i-rOOOJ •r-'a-ao 


O 3 


OJE-MC Or-ESoS-(US-SC0 


1 — •"-> 


otoo 3<:^ jzcu oun 


■a 


"O •>— O -O" 1 — S-c/5 x+JtO "OOJ 


ro ro 


Qjcooo i-T- EOTDCn E 




U1 'CcoOi — • 3'i- • •'(03<i;+J 


^ 3: 


3 «;}• O ro 3 C CO O^r— iX) • ■•- .SZ n3 


cr> Q. 


OSO-CUE -t-O OJ-CCI— 


3 1 


C T3 •<- CTl 3 >5r— (/) T3 • en Q E 


o -a 


EC-OCr— T3r— 1— C-r-3E O 


i- O) 


3rO<UE 30aj<U-"rO O-i- •--^ 


^ s- 


1— trt 3 O) r— o +-> cncM S- 1— ^ O 


+-) (1) 


0^3i— Olio rO-O |OS-jC O^ 


-M 


U+-> O-O a)E3ro° O-M "rOE 


n" r-* 


a>r— (J3-O^T-r— Lr)3 'ZS. 


•f— 


OJC-r- 1 — (U4->X(/1 C\JO"T3aL 


QJ 4- 


j=<uo<u</)+-> o OJ -i-ajs-^o 


Q. 


■Ml— CO.E (OOS-OJc— -t->i— lOO O 


>i C 


■Q 1— "O 3 4-> Q..C JD ro to O r — CO 


4J •!- 


S- C 3 OJ •'D Q.I— ro 0) (O O r— 




O'l-to +->-a-oro 1— Eo)Q.aj • 


(/) "O 


<4- 'l/irOCU OT3 -(-)0</> 


3 (U 


ETD'-~3 ■i--t-> •<U~-...3C0-i- ro 


s- -a 


<U CJ Cf^-"-r>:i:i — ro -— ~ O) O ■ — ro i- 01 s 


•1- c 


E ra(.-oQ.Q. '(/i-Ctosajc: 


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30(/)^faiQ.E'-3IE +JT-1. 


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>— r— T3rOE'ia.CU— «i— (/)0 


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r— 3 


>V)C<UO.S-T3i — cCOl— OLl. O" 


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lO'i-t— 1 CUCO o—IO •! -O-r- 


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(USM-Xl-O-MrOUOJ— ^Ln +J •.— 


C 


S- (OOJi — OJ CO«(0(/)CO 


l|_ OJ 


OC(/)l— S-T-.— (U_J<U OOTS- O) 


o x: 


Q-E'>- aj<4-E-J= oi"o 3<unrcn 


2 


3+J<U+J — -)->4->T3S^>^<4-+Ja.T3 


+-> 


(Ui— i/)a)i— ojrD s_3cr> -i-r— 3 


C 1— 


C:03</)T-.CLi_00r— .4JS-'i-Oi— 


q; -i- 


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00 




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LU 




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147 



(L"i/ndd £0L X) pa:inL9 snjiAOLiOd 




{9iun[0A auod l^^e^ :^g paiLdde uaaq 
6uLAeq njd Lno:t io % aA!.:;BLniunD sb pessaadxa) pa:in[3 snjiAoiLOd 



148 



with the adsorption of poliovirus to sludge and soil particles. Fur- 
thermore, Figure 4-7 shows that the passage of ten pore volumes 
(2250 ml) of sludge diluted with sludge liquor brought to a final CaCl2 
concentration of 0.01 N resulted in the gradual increase in the soil 
solution pH from 5.8 to the pH of the leaching solution, 8.3. Thus, 
the soil was unable to buffer the pH and this increase in pH into the 
basic range further prevented viral adsorption to the soil. 

Eustis fine sand . Experiments involving poliovirus suspended 
in sludge liquor were also performed using Eustis fine sand columns. 
The sludge liquor employed in these studies was produced by centrifug- 
ing lagoon sludge (LAG— see Table 3-2; 2/3 anaerobically digested and 
1/3 aerobically digested sludge), and subsequently passing the result- 
ing supernatant through a series of 0.45 and 0.25 urn Filterite filters 
and then adjusting the sludge liquor to pH 8.0. The filtration pro- 
cedure did not remove all bacterial cells from the sludge liquor. 
This was confirmed by microscopic examination of the sludge liquor at 
a magnification of lOOOx. Figure 4-8 shows that poliovirus break- 
through occurred at the first pore volume, and by the tenth pore 
volume, 100% of the applied virus had appeared in the leachate from 
a 10-cm column of this soil. Further research by Overman et al^. (un- 
published data) has confirmed that this sludge liquor strongly inter- 
feres with the adsorption of poliovirus type 1 and echovirus type 4 
to the Eustis fine sand. Moreover, these investigators demonstrated 
that this sludge liquor was not able to elute substantial numbers of 
previously adsorbed viruses (i.e., poliovirus and echovirus). 



149 



The mechanisms(s) of sludge liquor interference with virus 
adsorption to soils is (are) not well understood. It is known that 
water-soluble "humic substances" interfere with the sorptive capacity 
of soil and sediments toward viruses. The decrease in virus retention 
is due to humic fractions with a molecular weight of less than 50,000 
(Bitton et al_. 1977; Scheuerman et al . 1979). Anaerobically digested 
sludge contains fulvic acid fractions (Baham e_t al_. 1978; Holtzclaw 

et al. 1976, 1978; Sposito and Holtzclaw 1977; Sposito et al- 1976, 

+2 
1978) that are known to complex Ca ions, an important and ubiquitous 

metal cation in soil solution (Sposito et al^. 1978). This complexa- 
tion phenomenon may aid in the inhibition of virus adsorption to soils. 
Other mechanisms are probably involved in this inhibition process. 
When poliovirus was suspended in sludge liquor that had been filtered 
through a 0.22 \m Millipore filter, the percent breakthrough of this 
virus in Eustis fine sand columns was significantly reduced (i.e., 
4%) as compared to the breakthrough (34%) following the application of 
Filterite- treated sludge liquor (Overman et al_. , unpublished data). 
Microscopic examination showed that Filterite- treated sludge liquor 
contained substantial numbers of bacterial cells whereas no bacteria 
were found in Millipore- treated sludge liquor. It is then possible 
that these bacterial cells may compete with viruses for adsorption 
sites on the surface of the soil particles. This is a mere speculation 
that needs to be demonstrated under more controlled conditions. Com- 
petitive adsorption between viruses and bacteria has not been reported 
in the literature. 



150 



The sludge liquor employed in these experiments represents 
an artificial system probably never encountered in the environment. 
However, under field conditions, rain water may leach the sludge 
liquor from the applied sludge, which then may affect virus transport 
through the soil. The implications of this possible phenomenon in 
field situations deserve further investigation. 

Poliovirus Suspended in Undiluted 
Anaerobically Digested Sludge 

The movement of sludge-associated viruses through soil was 
next studied under more realistic conditions. Poliovirus was added 
to undiluted anaerobically digested sludge while stirring the mixture 
on a magnetic stirrer. One inch (2.5 cm) of seeded sludge was then 
applied on top of undisturbed cores of Eustis fine sand and Red Bay 
sandy loam. The sludge was allowed to soak in (in one experiment the 
sludge was allowed to air dry for 24 hours) and then worked under. 
The soil cores were then eluted with either 0.01 N CaCl^ or rain 
water. 

Red Bay sandy loam . Undisturbed soil cores of Red Bay sandy 
loam were treated with poliovirus-seeded sludge as described above. 
The applied sludge was allowed to air dry for 24 hours, under field 
conditions, before being mixed with the top 2.5 cm of soil to simulate 
field practices. The soil columns were then eluted with three to four 
pore volumes of rain water. Table 4-15 shows that there was a virus 
breakthrough in the second or the first pore volume collected. This 
virus breakthrough represented 0.1 and 0.2% of the total PFU applied to 



151 



TABLE 4-15. 



Retention of poliovirus type 1 by undisturbed cores of 
Red Bay sandy loam follov/ing the application of 2.5 cm 
of seeded anaerobically digested sludge (air dried 24 
hrs) and the subsequent elution with rain water 



No. of 
pore . 
volumes' 
eluted 



Poliovirus 

eluted 
(total PFU) 



% of Total 
PFU applied^ 
(cumulative) 



Conductivity 
of pore volume 
collected 
(pmho/cm 
at 25°C) 



pH of pore 

vol ume 
collected 



Column 1 



1/3 










245 


2/3 










195 


1 










125 


1 1/3 










106 


1 2/3 


1.1 


X 10^ 


0.2 


80 


2 







0.2 


82 


2 1/3 







0.2 


77 


2 2/3 







0.2 


72 


3 







0.2 


66 


3 1/3 







0.2 


66 


Column 2 











4.4 
4.7 
4.8 
5.1 
5.2 
5.1 
5.1 
5.0 
5.0 
5.0 



1/3 

2/3 

1 

1 1/3 

1 2/3 
2 

2 1/3 

2 2/3 
3 

3 1/3 



5.2 X 10' 





















0.1 
0.1 
0.1 
0.1 
0.1 
0.1 
0.1 
0.1 
0.1 
0.1 



232 

177 

157 

115 

98 

87 

75 

69 

62 

58 



4.8 
5.2 
5.5 
6.0 
5.6 
5.4 
5.6 
5.5 
5.4 
5.5 



TABLE 4-15. Continued. 



152 



No. of 

pore 

volumes^ 

eluted 


Poliovirus 

eluted 
(total PFU) 


% of Total, 
PFU applied^ 
(cumulative) 


Conductivity 
of pore volume 
collected 
(pmho/cm 
at 25°C) 


pH of pore 

volume 
collected 


3 2/3 









0.1 


54 


5.6 




4 









0.1 


54 


5.7 




4 1/3 









0.1 


49 


6.4 





One pore volume for these cores equals 471 ml. The undisturbed 
soil cores were 54 cm in length and 5.0 cm internal diameter; consists 
of the Al , A2, Bit and B21t horizons of the Red Bay sandy loam (see 
Table 4-3). The cores were not conditioned. 

One inch or 2.5 cm (51.6 ml) of anaerobically digested sludge 
(GDAN— see Table 3-2 ; solids content, conductivity and pH equal to 
2.0%, 3250 umho/cm at 25°C and 8.3, respectively) seeded with a total 
of 5.1 X 10^ PFU of poliovirus was applied to each of the cores. The 
cores were then placed on the roof of the Environmental Engineering 
Sciences building at the University of Florida, Gainesville. The ap- 
plied sludge was allowed to air dry for 24 hrs and then was worked 
under 2.5 cm. Elution with rain water was subsequently undertaken. 
This solution was applied from an inverted, self-regulated, 1 liter 
Erlenmeyer flask set to maintain a 2.5 cm hydraulic head on the cores. 
The flow rate through the cores was measured at 2.4 ml/min. The rain 
water was collected next to the Environmental Engineering Sciences 
building. See Table 4-2 for chemical characteristics of the rain 
water. 



153 



the soil columns. Afterwards, no virus was detected in the leachates, 
even after the passage of 77.5 to 93 cm of rain (equivalent to 1,570 
to 1,884 ml of rain water). The observed breakthrough of poliovirus 
when the first pore volume had percolated through column 2 is not 
surprising due to the fact that the columns were not initially satur- 
ated. A soil column treated with 2.5 cm of sludge and subsequently 
eluted with 0.01 N calcium chloride was used as a control. From 
Table 4-16, it is clear that no virus could be detected in the column 
leachates. As discussed earlier, the presence of calcium chloride in 
. the soil solution readily enhances virus adsorption to the soil matrix. 
However, rain water was able to transport 0.1 to 0.2% of the total 
applied viruses through the soil profile. This breakthrough would 
probably be lower if the soil was. allowed to dry for a longer period 
of time under field conditions (see Chapter V), 

The results presented above show that the Red Bay sandy loam 
studied is effective in retaining viruses during sludge application. 
Virus associated with sludge solids will be retained at the surface 
of the soil matrix and will be inactivated with time due to environ- 
mental factors (e.g., temperature, drying and solar radiation). In 
Table 3-6, the adsorption of poliovirus to anaerobic sludge solids 
during a 12-hour contact period is found to range from 52.8% to 69.1%. 
Thus, the effectiveness of virus retention by soils during sludge 
application is partly attributed to the capacity of sludge solids to 
bind viruses in the top of the soil profile. However, viable "free" 
virus (i.e., viruses not associated with sludge solids or dissociated 



154 



TABLE 4-16. Retention of poliovirus type 1 by an undisturbed core of 
Red Bay sandy loam following the application of 2.5 cm of 
seeded anaerobically digested sludge and the subsequent 
elution with 0.01 N CaCl2 



No. of Poliovirus % of Total, 

pore eluted PFU applied 

volumes^ (total PFU) (cumulative) 
eluted 



1/3 

2/3 

1 
11/3 
12/3 

2 
2 1/3 

2 2/3 

3 
3 1/3 

3 2/3 

4 
4 1/3 

^One pore volume for this core equals 471 ml. The undisturbed 
soil core was 54 cm in length and 5.0 cm internal diameter; consists of 
the Al , A2, Bit and B21t horizons of the Red Bay sandy loam (see 
Table 4-3). The core was not conditioned. 

One inch or 2.5 cm (51.6 ml) of anaerobically digested sludge 
(GDAN~see Table 3-2; solids content, conductivity and pH equal to 
2.0%, 3250 umho/cm at 25°C and 8.3, respectively) seeded with a total 
of 2.2 X 10^ PFU of poliovirus was applied to the core, allowed to 
soak in and then, worked under 2.5 cm. Elution with 0.01 N CaCl2 
(conductivity and pH equal to 1210 ymho/cm at 25°C and 6.4, respectively) 
was subsequently undertaken. This solution was applied from an in- 
verted, self-regulated, 1 liter Erlenmeyer flask set to maintain a 
2.5 cm hydraulic head on the core. The flow rate through the core was 
measured at 3.5 ml/min. 



Conductivity 
of pore volume 
collected 
(ymho/cm at 
25°C) 


pH of pore 

volume 
collected 


650 


4.5 


1360 


4.5 


1570 


4.8 


1660 


5.2 


1720 


4.8 


1750 


5.0 


1760 


4.9 


1770 


4.9 


1770 


4.8 


1770 


4.8 


1770 


4.8 


1770 


4.8 


1770 


4.7 



155 



from sludge solids as a result of changes in the physico-chemical 
properties within the soil matrix--see Table 3-6) will move in the 
soil solution or be retained by the soil particles as govered by pH, 
flow rate, conductivity, and the presence of soluble organic materials 
(Bitton 1975; Gerba et al_. 1975). Of particular importance in the 
retention of these "free" viruses by the soil is the nature of the 
soil itself and, more specifically, the clay content of the soil. Due 
to their large surface area, the clay minerals in soils comprise the 
fraction most active in retaining viruses (Carlson et al_. 1968). The 
Red Bay sandy loam studied displayed a clay content ranging from 13.6% 
in the Al horizon to 36.2% in the B21t horizon (see Table 4-3). The 
dominant clay in the Al, A2, and Bit horizons was vermiculite, while in 
the B21t horizon, it was gibbsite. The retention of "free" poliovirus 
during sludge application to the Red Bay sandy loam is attributed to 
the adsorptive capacity of the clay fraction found throughout the soil 
profile and accumulated in the deeper horizons (e.g.. Bit and B21t). 
Moreover, this soil contained iron oxides which are also effective in 
retaining viruses (Bitton 1980a). The interaction between iron oxides 
and viruses in soils deserves further study. 

Eustis fine sand . Similar application of sludge to an undis- 
turbed core of Eustis fine sand and subsequent elution with rain water 
resulted in the breakthrough of 29.3% of the total (5.7 x 10^ PFU) 
poliovirus applied (see Figure 4-9). A peak in conductivity (400 
umho/cm at 25°C) was found at the 1.5 pore volume and this was probably 
due to sludge leachates passing through the soil matrix. This 



FIGURE 4-9. Movement of poliovirus type 1 through an undisturbed 
core of Eustis fine sand ( conditioned with rain water ) 
following the application of 2.5 cm of seeded anaerobi- 
cally digested sludge and the subsequent elution with 
rain water 

One pore volume for the core used equals 234 ml. 
The undisturbed soil core was 33 cm in length and 
5.0 cm internal diameter; consists of the Ap and 
A21 horizons of the Eustis fine sand (see Table 
4-3). The core was initially conditioned with 
5 pore volumes of rain water. One inch or 2.5 cm 
(51.6 ml) of anaerobically digested sludge (GDAN— 
see Table 3-2; solids content, conductivity and 
pH equal to 2.0%, 3250 ymho/cm at 25°C and 8.3, 
respectively) seeded with a total of 5.7 x 10^ 
PFU of poliovirus was applied to the core, allowed 
to soak in and then, was worked under 2.5 cm. 
Elution with rain water was subsequently under- 
taken. This solution was applied from an inverted, 
self-regulated, 1 liter Erlenmeyer flask set to 
maintain a 2.5 cm hydraulic head on the core. The 
flow rate through the core was measured at 3.9 
ml/min. The rain water was collected next to the 
Environmental Engineering Sciences building at the 
University of Florida, Gainesville. See Table 4-2 
for chemical characteristics of the rain water. 



157 



33 
30 
27 
24 
21 

18 
15 
12 



6 

3 




1 





-5.0 



-^-ryy-crc^ tr-y-o 



1 



3 4 5 6 7 

No. of pore volumes eluted 



10 



158 



undisturbed core was initially conditioned with rain water. When an 
identical, undisturbed core was conditioned with 0.01 N CaCl2. only 
10.6% of the total poliovirus applied was detected in the effluent 
by the 8.5 pore volume (see Figure 4-10). Conditioning with calcium 
chloride enhanced the adsorption of viruses to the soil. Thus, it was 
found that the nature of the conditioning solution can affect the virus 
breakthrough pattern later obtained. 

It appears then that Eustis fine sand, under saturated flow, 
does not retain viruses effectively (in Chapter V,data are presented 
on virus transport through Eustis fine sand cores under unsaturated 
flow and under field conditions). There is a dramatic difference 
between the virus "retention potential" of the Red Bay sandy loam 
(99.8%) and the Eustis- fine sand (70.7%). It is postulated that the 
low adsorptive capacity of the Eustis fine sand is a direct result of 
its low clay content (only 3.2%, average for the Ap and A21 horizons, 
see Table 4-3). In the case of the Eustis fine sand, sludge applica- 
tion could lead to ground water contamination with pathogenic viruses. 
However, the results presented for the Red Bay sandy loam indicate 
that, under appropriate conditions, sludge application could be 
undertaken without threatening the quality of ground water with 
viruses. 

Condi tioned-Dewatered Sludge 

At many wastewater treatment plants around the country, 
digested sludges are conditioned with polymers prior to dewatering. 
At the Main Street wastewater treatment plant in Gainesville, Florida, 



FIGURE 4-10. Movement of poliovirus type 1 through an undisturbed 

core of Eustis fine sand ( conditioned with 0.01 N CaCl2 ) 
following the application of 2.5 cm of seeded anaerobi- 
cally digested sludge and the subsequent elution with 
rain water 

One pore volume for the core used equals 234 ml. 
The undisturbed soil core was 33 cm in length and 
5.0 cm internal diameter; consists of the Ap and 
A21 horizons of the Eustis fine sand (see Table 
4-3). The core was initially conditioned with 
5 pore volumes of 0.01 N CaCl2 (conductivity and 
pH equal to 1210 pmho/cm at 25°C and 6.4, respec- 
tively). One inch or 2.5 cm (51.6 ml) of anaerobi- 
cally digested sludge (GDAN--see Table 3-2; 
solids content, conductivity and pH equal to 
2.0%, 3250 ymho/cm at 25°C and 8.3, respectively) 
seeded with a total of 9.8 x 10^ PFU of poliovirus 
was applied to the core, allowed to soak in and 
then, was worked under 2.5 cm. Elution with rain 
water was subsequently undertaken. This solution 
was applied from an inverted, self -regulated, 
1 liter Erlenmeyer flask set to maintain a 2.5 cm 
hydraulic head on the core. The flow rate through 
the core was measured at 3.9 ml/min. The rain 
water was collected next to the Environmental 
Engineering Sciences building at the University 
of Florida, Gainesville. See Table 4-2 for 
chemical characteristics of the rain water. 



160 




2 3 4 5 6 7 
No. of pore volumes eluted 



161 



for example, digested sludge is conditioned with a cationic polymer 
(Hercofloc #871, Hercules Co., Atlanta, Ga.) and subsequently dewatered 
by centrifugation. The fate of poliovirus seeded in anaerobically 
digested sludge was determined following conditioning and dewatering 
of sludge as described above. As shown in Table 4-17, in two experi- 
ments, 72.2% and 89.3% of poliovirus initially added to the sludge was 
found in the conditioned-dewatered sludge. Moreover, in both experi- 
ments, 99.9% of the virus recovered from the conditioned-dewatered 
sludge was found associated with the sludge solids (see Table 4-18). 
The application of the conditioned-dewatered sludge containing polio- 
virus to columns of Red Bay sandy loam and the subsequent elution 
with rainwater did not result in any virus breakthrough (see Table 
4-19). These results show that viruses present in conditioned- 
dewatered sludge are effectively retained by soils. 

Chemical Sludges 

In addition to biological sludges, chemical sludges may also 
be produced during wastewater treatment. These sludges may be produced 
during primary treatment when coagulation (using alum, ferric chloride, 
or lime) is combined with sedimentation to upgrade the removal 
efficiency of the treatment process (i.e., intermediate treatment) or 
during advanced wastewater treatment (tertiary treatment) (Malina et a^. 
1976). Previous research has demonstrated that viruses are concen- 
trated in alum and ferric chloride sludges (Lund and R0nne 1973; Wolff 
et al_. 1974). However, in lime sludges, viruses have been found to be 
effectively inactivated (Lund and R0nne 1973; Sattar et £!_. 1976). 



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163 



TABLE 4-18. Association between poliovirus type 1 and conditioned- 
dewatered sludge solids 



Experiment Virus in Virus in Viable Solids- 
no. unfractionated sludge unadsorbed*^ associated^ 
sludge"^ supernatant^ virus virus 
(total PFU) (total PFU) (%) (%) 

1 2.5 X 10^ 1.1 X 10^ 0.04 99.9 

2 2.6 X 10^ 3.1 X 10^ 0.1 99.9 



^The sludge solids were not separated prior to assaying. 

The methods used to produce the dewatered sludges and to determine 
the amount of viruses present in the dewatered sludges are described in 
Table 4-17. 

^The sludge was clarified by centrifugation at 1400 x g for 10 min 
at 4°C and the supernatant was subsequently assayed. 

•^The "viable unadsorbed virus (%)" values were calculated as shown 
in the Materials and Methods section. 

®The "solids-associated virus (%)" values were estimated as shown 
in the Materials and Methods section. 



164 



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165 



Experiments were undertaken to assess the capacity of the Red Bay 
sandy loam subsoil to retain viruses following the application of 
chemical sludges (alum, ferric chloride, and lime). The aim of this 
research was to determine if there is a risk of groundwater contamina- 
tion with viruses when chemical sludges are disposed on land. 

The chemical sludges (alum, ferric chloride, and lime) were 
precipitated from poliovirus-seeded, raw sewage. As shown in Table 
4-20, variable fractions of the seeded viruses were recovered from 
the chemical sludges produced (22.6% to 95.6% for alum sludges, 
9.8% to 13.3% for ferric chloride sludges, and 0% to 0.1% for lime 
sludges). Since the viruses became embedded in the sludges produced 
during the flocculation process, it is likely that not all viruses 
present in the sludges were recovered. It is generally believed that 
embedded viruses are difficult to elute. It does appear, however, 
that due to the high pH of the lime sludges, most of the viruses 
originally seeded in the raw sewage were inactivated (see Tables 
4-20 and 4-21). The association between poliovirus and chemical 
sludge solids was then evaluated. It was found that from 97% to 100% 
of input virus was associated with alum, ferric chloride, and lime 
sludge solids (Table 4-21). In one sample (experiment no. 2) of 
lime sludge, all viruses were inactivated due to the high pH (pH 11.3) 
generated during the process. The application of these virus-seeded 
sludges to soil columns of Red Bay sandy loam did not result in any 
virus breakthrough following leaching with two to ten pore volumes 
of rainwater (Table 4-22). It is worth stressing that no virus could 



"J»»' 



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167 



TABLE 4-21. Association between poliovirus type 1 and chemical sludge 
solids 



Sludge^ Experiment 
type no. 



Virus in 



unfractionated^ 



Virus in Viable Solids- 
sludge unadsorbed^ associated 
sludge supernatant^ virus virus 
(total PFU) (total PFU) (%) {%) 



Alum 


1 


1.2 


X 


10^ 


3.6 


X 


10^ 


3.0 


97.0 




2 


4.3 


X 


10^ 


1.5 


X 


10^ 


0.3 


99.7 




3 


1.1 


X 


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5.0 


X 


10^ 


0.5 


99.5 


Ferric 


1 


4.7 


X 


10^ 


6.5 


X 


10^ 


1.4 


98.6 


chloride 


2 


4.4 


X 


10^ 


1.2 


X 


105 


2.7 


97.3 




3 


5.2 


X 


10^ 


7.0 


X 


10^ 


1.3 


98.7 


Lime 


1 


7.5 


X 


10^ 












100.0 




2 























^The chemical sludges were precipitated from virus-seeded, raw sewage. 
The methods used to produce these sludges and to determine the amount of 
viruses present in the sludges are described in Table 4-20. 

The sludge solids were not separated prior to assaying. 

^The sludge was clarified by centrifugation at 1400 x g for 10 min at 
4°C and the supernatant was subsequently assayed. 

The "viable unadsorbed virus (%)" values were calculated as shown in 
the Materials and Methods section. 

^The "solids-associated virus (%)" values were estimated as shown in 
the Materials and Methods section. 



168 



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170 



be detected in any of the soil leachates despite the concentration of 
the leachates by membrane filtration (160-fold concentration). 
Lime-Stabilized, Chemical Sludges 

Large quantities of chemical sludge are usually produced during 
primary treatment and may be stabilized with the use of lime (Farrell 
et aj_. 1974). The effect of lime stabilization on the infectivity of 
poliovirus present in chemical sludges (alum and ferric chloride ) was 
investigated next. The fate of poliovirus following the application of 
lime-stabilized, chemical sludges to soil columns was also studied. 

The stabilization of chemical sludges (alum and ferric chloride 
sludges) with lime resulted in almost comolete inactivation of polio- 
virus (Tables 4-23 and 4-24). It was, thus, not surprising to observe 
that the virus did not break through when the stabilized sludges were 
applied to soil columns of Red Bay sandy loam which were subsequently 
leached with ten pore volumes of rainwater (Table 4-25). 

These results show that lime stabilization of chemical sludges 
can effectively inactivate viruses. Moreover, as in the case of 
conditioned-dewatered sludge and chemical sludges, viruses remaining 
in lime-stabilized, chemical sludges can be effectively retained by soils 
following sludge disposal on land. 
Effect of Soil Bulk Density on Poliovirus Transport 

The effect of soil bulk density on poliovirus transport was studied 
using laboratory-packed soil columns of Red Bay sandy loam subsoil. The 
air-dried soil was packed into IQ-cm acrylic plastic columns at bulk 
densities of 1.45 and 1.60 g/cm^. Poliovirus was suspended in primary 



171 



TABLE 4-23. Inactivation of poliovirus type 1 following lime stabilization 
of chemical sludges 



Sludge^ 
type 


Concentration 

of 

lime° used 

(mg/1) 


pH, 30 min 
after the 
addition 
of lime 


Virus in un- 
fractionated^ 

sludge, 

before 

liming 


Virus in 
unfractionated 
sludge, 30 min 
after liming 




Total PFU 


Total PFU 


Recovery 

(%) 


Alum^ 

Ferric 
chloride® 


1389 
625 


11.3 
11.1 


1.1 X 10^ 

5.2 X 10^ 




6.2 X 10^ 



0.1 



The chemical sludges were precipitated from virus-seeded, raw sewage. 
The methods used to produce these chemical sludges and to determine the 
amount of viruses present in the sludges are described in Table 4-20. 

'^An aqueous slurry of lime (5% Ca(0H)2) was added to the chemical 
sludges shown until a pH of 11.5 was achieved and maintained for 5 min. The 
final concentrations of Ca(0H)2 used arppear in the table above. A contact 
time of 30 min was allowed while mixing the suspension on a magnetic stirrer. 

^The sludge solids were not separated prior to assaying. 

^Percent recoveries shown were calculated based on the corresponding 
unfractionated sludge assay before liming as 100%. 

®The lime-stabilized, chemical sludges were applied to columns of Red 
Bay sandy loam subsoil (see Table 4-25). 



172 



TABLE 4-24. Association between poliovirus type 1 and lime-stabilized, 
chemical sludge solids 



Sludge^ 
type, 
lime- 
stabilized 



Virus in , 
unfractionated 
sludge 
(total PFU) 



Virus in 
sludge 
supernatant^ 
(total PFU) 



Viable 
unadsorbed" 
virus 

(%) 



Sol ids - 

associated^ 

virus 

(%) 




The chemical sludges were precipitated from virus-seeded, raw 
sewage. The methods used to produce these sludges and to determine the 
amount of viruses present in the sludges are described in Table 
The chemical sludges were then stabilized with Ca(0H)2 as described in 
Table 4-23. 

The sludge solids were not separated prior to assaying. 

''The sludge was clarified by centrifugation at 1400 x g for 10 min 
at 4°C and the supernatant was subsequently assayed. 

The "viable unadsorbed virus (%)" values were calculated as shown 
in the Materials and Methods section. 

^The "sludge solids-associated virus (%)" values were estimated as 
shown in the Materials and Methods section. 



173 



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x: r- E r- d; .— 


o +j x: •!- 03 




T3 


O CO--- o 


h— tOCU03CtO+JXI 


t/1 T- 3 1 — +J 




CJ -r- 


rO • (U O 


-Q x: o o c o 03 


o > to o to 




•r- S- 


^ CD 


CU O -r- -r- E dJ 1— 


•1- a. 1 


E 


S- O 


-a -a 


to E •+-> 3 r- 


+-> CU CU 


3 


i- 1 — 


-a 3 CU 


CU dl CU 3 1 — 1 — OJ 


o S- i. E 


r— 


CU .£: 


c 1— (o 


x: x: jz r— o o d) 


d) d) O •!- 


cC 


Ll_ u 


03 (O 3 


+J t— O LtJ O O C/1 


4-3 4-.— 



174 



wastewater effluent (pH of 7.5 and conductivity of 340 pho/cm at 25°C) 
and applied continuously to the soil columns at approximately 3.5 ml/min 
using a peristaltic pump. As shown in Figure 4-11, poliovirus moved 

faster and appeared in the leachates in greater numbers in columns 

3 
packed at a bulk density of 1.45 g/cm (i.e., columns 1 and 2) than in 

3 
columns packed at a bulk density of 1.60 g/cm . Only column 3 at a 

3 
bulk density of 1.45 g/cm displayed slow movement through the soil 

3 
typical of the soil columns at a bulk density of 1.60 g/cm (see 

Figure 4-11). It appears that in the columns displaying large viral 
breakthroughs (i.e., columns at bulk density of 1.45 g/cm ) , the 
virus-seeded primary effluent followed a less circuitous path through 
the soil resulting in less opportunity for poliovirus adsorption to 
the soil particles. In spite of this, however, no significant 
statistical difference was found between the fractions of polio- 
virus eluted at the tenth pore volume in soil columns packed at the 
2 different bulk densities (see Table 4-26). 



FIGURE 4-11. Movement of poliovirus type 1 suspended in primary 
wastewater effluent through 10-cm columns of Red Bay 
sandy loam subsoil packed at bulk densities of 1.45 
and 1 .60 g/cm3 

One pore volume for the columns packed at a bulk 
density of 1.45 or 1.60 g/cm^ equals 80 or 70 ml, 
respectively (see Table 4-5). The laboratory- 
packed columns were 10 cm in length and 4.8-cm 
internal diameter. The sample of Red Bay sandy 
loam subsoil used consisted mainly of the A2 and 
Bit horizons (see Table 4-3). The columns were 
conditioned with 2 pore volumes of nonseeded 
primary wastewater effluent. Poliovirus was then 
suspended in the primary wastewater effluent at 
the concentrations shown in the figure in paren- 
theses and applied to the columns. All solutions 
were applied continuously to the columns at 
approximately 3.5 ml/min using a peristaltic pump 
(Buchler, Fort Lee, N.J.). The primary wastewater 
effluent sample used displayed a pH of 7.5 and a 
conductivity of 340 ymho/cm at 25°C. The mean pH 
and conductivity (ranges indicated by vertical 
lines) of each pore volume were calculated from 
the individual values obtained for the 6 columns. 



176 



12 



=3 




U. 




a. 




p— 




« 




•M 




O 




■M 






«— «^ 


4- 


<u 


O 


E 




3 


^ 


r— 




O 


<U 


> 


> 






dJ 


4J 


s- 


fO 


o 


r— 


Q. 


3 




E-C 


3 


U 


U 


(O 




(U 


W) 




<T3 


•)-> 




(O 


-a 




<U T3 


(/) 


0) 


1/1 


•r" 


0) 


^— 


s- 


Q. 


CL Q. 


X 


(0 


0) 




*._• 


C 




0) 


■a 


cu 


OJ J2 


4J 




=J 


en 


r-" 


c 


0) 


•r" 




> 


t/i 


(O 


3 


^ 


S- 




•f" 




> 




o 





— I \ 1 1 1 — 

Bulk density 1.45 g/cm^ 
- 9- # Column 1 

(2.7 X 103 PFU/ml) 

O O Column 2 

(2.1 X 10-^ PFU/ml) 

O Column 3 

(2.1 X 1Q3 PFU/ml) 
.Bulk density 1.60 g/cm>^ 

%- -• Column 4 

(2.9 X 103 PFU/ml) 

. O O Column 5 

(2.9 X 103 PFU/ml) 

Column 6 

(2.9 X 103 PFU/ml] 




3 4 5 6 7 
No. of pore volumes eluted 



10 



177 



TABLE 4-26. Total amount of poliovirus type 1 detected in ten pore 
volumes of leachate from 10-cm columns of Red Bay sandy 
loam subsoil packed at bulk densities of 1.45 and 1.60 
g/cm3 



Bulk density Column no. At pore volume no. 10 

Poliovirus Poliovirus Mean'- poliovirus 

applied breakthrough breakthrough for 

(total PFU) (%)'^ each bulk density 



(%b + SEd) 



1.45 1 2.2 X 10^ 12 7.3 + 2.9 

2 1.7 X 10^ 7.9 

3 1.7 X 10^ 2.0 

1.60 4 2.0 X 10^ 5.6 2.6 + 1.6 

5 2.0 X 10^ 1.6 

6 2.0 X 10^ 0.5 



Complete data and experimental procedures are shown in 
Figure 4-11 . 

Expressed as cumulative percent of total PFU applied at pore 
volume no. 10. 

^The two mean values shown are not significantly different at the 
0.05 level when subjected to a two-tailed,t-test. 

Abbreviation for standard error. 



-X ' 



CHAPTER V 

RETENTION AND INACTIVATION OF ENTEROVIRUSES 

IN SOIL CORES TREATED WITH VIRUS-SEEDED SLUDGE 

AND EXPOSED TO THE NORTH-CENTRAL FLORIDA ENVIRONMENT 



Introduction 
Although numerous studies have been conducted to determine the 
survival (Bagdasaryan 1964; Derbyshire and Brown 1978; Duboise et a^. 
1976; Green 1976; Lefler and Kott 1974; Hurst et al. 1980a, 1980b; Moore 
et al. 1977; Sobsey et al. 1980a; Yeager and O'Brien 1979a, 1979b) and 
transport pattern (Bitton et al_. 1976; Drewry and Eliassen 1968; 
Duboise et al_. 1976; Funderburg et al_. 1979; Gerba and Lance 1978; 
Greene 1976; Laak and McClean 1967; Lance and Gerba 1980; Lance et a^. 
1976; Landry etal. 1980; Lefler and Kott 1974; Robeck et al. 1962; 
Schaub and Sorber 1977; Scheuerman et al. 1979; Sobsey et al- 1980a; 
Young and Burbank 1973) of viruses following water or wastewater appli- 
cation to soils, few have been undertaken to assess viral persistence 
and transport in sludge- treated soils (see reviews by Bitton 1975, 
1978, 1979b, 1980a, 1980b; Duboise etal. '•979; Foster and Engelbrecht 
1973). Viruses have been found to be inactivated in sludge allowed to 
dry on the soil surface (Brown et al. 1980; Hurst et al. 1978; Nielsen 
and Lydholm 1980), Other investigators have demonstrated that, under 
cold winter temperatures, viruses can persist in sludge-amended soils 
for as long as six months (Tierney et al. 1977; Damgaard-Larsen et al. 
1977). Damgaard-Larsen et al. (1977) used lysimeters to study viral 

178 



T79 



transport in sludge- amended soils and noted that viruses were completely 
retained by the soils under study. 

In this chapter, the survival and transport of viruses in 
sludge- treated soils were evaluated under field conditions. Undisturbed 
soil cores were used and environmental parameters (i.e., soil tempera- 
ture, soil moisture, and rainfall) were monitored. The protocol of 
sludge disposal to soil was similar to that practiced at sludge dis- 
posal sites. Virus survival and transport were monitored during three 
different runs, using the same soil cores. 

Materials and Methods 
Viruses and Viral Assays 

Poliovirus type 1 (strain LSc) and echovirus type 1 (strain 
Farouk- prototype strain according to Wulff and Chin 1972) were used in 
the research reported in this chapter. Some general properties of 
echoviruses are shown in Table 5-1 (see Table 3-1 for general proper- 
ties of polioviruses). Stocks of echovirus were prepared as described 
for poliovirus in Chapter III (see page 53 ). Viral stocks were con- 
centrated by either ultracentrifugation or by the method developed by 
Farrah et al_. (1978) that involved blending with trichlorotrifluoro- 
ethane (Freon 113, DuPont DeNemours Co., Wilmington, Delaware) followed 
by concentration on Filterite filters (Filterite Corp., Timonium, Mary- 
land). The concentrated viruses were kept at -70°C until used. Echo- 
virus was assayed by the plaque technique as described for poliovirus 
in Chapter III (seepages 53-56 ). Each viral count shown represents 



1.8Q 



TABLE 5-1, General properties of echoviruses 



Property 



Value' 



Nucleic acid 

Molecular weight 

of nucleic acid (daltons) 

Particle diameter (ran) 

Morphology 

Stability at 4"'C 

Stability at pH 3.0 

Stability in ether 



RNA (single-stranded) 
2 X 10^ 

17 to 30 

Icosahedral 

Stable for 1 to 2 years 

Stable for 3 hours at 25°C 

Stable 



All data were obtained from Wulff and Chin (1972) 



181 



the average of triplicate counts. The numbers of viruses were expressed 
as plaque- forming units (PFU). 

Sludges 

Two sludge types were used in these experiments: aerobically 
digested sludge (GDA 180— see Table 3-2) sampled at the Main Street 
wastewater treatment plant of Gainesville, Florida, and lagooned sludge 
(LAG— see Table 3-2) sampled at the West Florida Agricultural Experiment 
Station, Jay, Florida. The lagooned sludge is a mixture of aerobically 
digested sludge (1/3) and anaerobically digested sludge (2/3) from the 
Montclair and Main Street wastewater treatment plants of Pensacola, 
Florida, respectively (see Table 3-2). The mixture was kept in a lagoon 
at the experiment station before ultimately being disposed of on land. 
The sludges were collected and sludge parameters (i.e., pH and solids 
content) were measured as described in Chapter III (see page 56). The 
sludge conductivity was measured using a Beckman conductivity bridge 
model RC 16B2 (Beckman Instruments, Fullerton, California). The sludges 
used were not autoclaved or decontaminated in any other way. 

Association of Seeded Viruses with Sludge Solids 

Poliovirus or echovirus stock in phosphate-buffered saline (PBS) 
containing 2% fetal calf serum (FCS) (see Appendix for more details 
on the composition of this solution) was added directly to sludge at 
the rate of 1 ml of virus stock per 1 ,000 ml of sludge and while stirring 
the suspension using a magnetic stirrer. Magnetic stirring was con- 
tinued for 10 to 60 minutes and then the association of viruses with 
sludge solids was determined using the procedure outlined in detail in 



182 



Chapter III(See page 62). Briefly, an aliquot of the unfractionated 
sludge (i.e., sludge sample without solids separated) was diluted 
in PBS containing 2% FCS and assayed directly for viruses by the 
plaque technique. This method (i.e., sludge dilution and subsequent 
direct assay on cell cultures) has been previously shown to be highly 
efficient in the recovery of poliovirus from unfractionated sludge 
(see Chapter III, page 62 and Table 3-3). Similar efficient 
recovery of echovirus from unfractionated sludge was also observed. 
The unfractionated sludge assay was performed in order to determine the 
total amount of virus present in the sludge sample. An aliquot of the 
sludge was subsequently centrifuged at 1 ,400 x g for 10 minutes at 4°C. 
The sludge supernatant produced was assayed for viruses. The "viable 
unadsorbed virus" and "sludge solids-associated virus" fractions were 
calculated as shown in Chapter III (see page 64 ), Following the 
. initial viral assays, virus-seeded sludge was applied to soil cores 
as described below. 

Soil 

The soil used in the research reported in this chapter was a 
Eustis fine sand sampled at the agronomy farm. University of Florida, 
Gainesville. This soil was classified as a Psammentic Paleudult, sandy, 
siliceous, hyperthermic (Calhoun et al. 1974). Some characteristics of 
this soil are shown in Table 4-3. The percent organic matter in this 
soil was measured at less than 1% (Calhoun et al_. 1974). 



183 



Fate of Viruses in Soil Cores 

The survival and transport (i.e., movement or retention) of 
poliovirus and echovirus in soil cores treated with virus-seeded 
sludge was studied under natural conditions. 

Undisturbed soil cores . Undisturbed soil cores (Blake 1965; 
Sanks et aj_. 1976) of Eustis fine sand were used and they were 
obtained by driving polyvinyl chloride pipes into the soil at the 
agronomy farm. University of Florida, Gainesville, as described in 
Chapter IV (see pages 102-103). The undisturbed soil cores were 
obtained in a manner that preserved the natural structure and bulk den- 
sity of the soil as found in the field (see Table 4-3). The soil cores 
were 33 cm in length (pipes were 40 cm in length) and had an internal 
diameter of 5.0 cm (small soil cores) or 15.5 cm (large soil cores), 
and thereby consisted of the Ap and A21 horizons of the fine sand 
(see Table 4-3). Two small soil cores and four large soil cores were 
employed. A polypropylene screen (105-pm pore size) which supported 
the soil while allowing the free movement of water and viruses (i.e., 
did not adsorb viruses in soil leachates), and a spout were secured 
at the bottom of each small soil core. Porous ceramic cups attached to 
spouts, on the other hand, were installed at the bottom of the large 
soil cores. The porous ceramic cups used were 6.9 cm long, and had a 
wall thickness of 0.23 cm and a pore diameter of 1.4 to 2.1 ym (no. 
2131, Soil Moisture Equipment Corp., Santa Barbara, California). These 
cups restricted the movement of water somewhat and consequently produced 
an artificial groundwater table in the bottom part of the soil during 



184 



periods of rainfall. Similarly, Robeck et al_. (1962) simulated a 
groundwater table in a sand column that was sealed at the bottom. 
The capacity of the porous ceramic cups to retain viruses was evaluated 
as described below. All six soil cores were exposed to natural condi- 
tions outside the Environmental Engineering Sciences building at the 
University of Florida, Gainesville. The soil cores rested on a wooden 
box such that soil leachates produced during natural rainfall could be 
collected (see Figure 5-1). Unlike the small soil cores, the large soil 
cores were insulated by surrounding them with duct insulation as shown 
in Figure 5-1. All soil cores were treated with virus-seeded sludge 
as described below. 

Porous ceramic cups . The retention capacity of porous ceramic 
cups towards viruses was evaluated using poliovirus suspended in a rain 
leachate from a small undisturbed core (see above) of Eustis fine sand. 
The rain leachate was produced in the laboratory by passing rain water 
continuously through the small soil core. The rain water was applied 
from an inverted, self-regulated, 1-liter Erlenmeyer flask set to main- 
tain a 2.5-cm hydraulic head on the soil core (Sanks et aj_. 1976). 
Leachate from the soil core (400 ml) was seeded with poliovirus. The 
virus-seeded leachate was then divided into two fractions of 200 ml and 
each fraction was passed through a sterile ceramic cup (no. 2131~see 

characteristics above) with the use of a vacuum pump at a flow rate of 

-1 -2 
approximately 2 ml hr cm . The concentration of poliovirus in rain 

leachate was determined before and after passage through the porous 

ceramic cups in order to claculate the percent retention of the virus. 



FIGURE 5-1. Photograph of the soil cores of Eustis fine sand used 
in this study 

Details on the procedures used to prepare these 
soil cores appear on pages 183 to 184. Four large 
soil cores (i-e., LCI through LC4) and two small soil 
cores (i.e., SCI and SC2) were employed as seen 
in the photograph. 



186 




187 



As shown in Table 5-2, 38.7% (mean for the two ceramic cups) of polio- 
virus suspended in rain leachate was lost (presumably retained by 
ceramic cups) during passage through the porous ceramic cups. Other 
investigators have found substantially greater retention of poliovirus 
type 1 by similar porous ceramic cups (i.e., 75% to 99.7%) when the 
virus was seeded in dechlorinated tapwater or in unchlorinated activated 
sludge effluent (Sobsey 1976; Wang et al_. 1980b). These solutions 
displayed greater conductivity values [e.g., 580 ymho/cm for tapwater 
and 787 ymho/cm for activated sludge effluent (Wang et al_. 1980b)] than 
that found for the rain leachate (i.e., 18 ymho/cm—see Table 5-2) 
employed in this study. Consequently, the abundantly present salts 
in tapwater and activated sludge effluent promoted greater viral 
adsorption to the ceramic material. Wang et al_. (1980b) also demon- 
strated that echovirus type 1 (strain V239~isolated from groundwater) 
seeded in tapwater or activated sludge effluent was retained (i.e., 
30% to 86%) by ceramic cups but to a lesser degree than observed for 
poliovirus type 1. Since the strain of echovirus type 1 used was 
previously shown to adsorb poorly to soil, it is likely that this 
virus did not adsorb efficiently to the ceramic material either (Wang 
et al. 1980b). In addition to viruses, fecal coliforms in water have 
also been reported to be retained by ceramic cups (Dazzo and Rothwell 
1974). It is worth noting that the rain leachate used herein to 
evaluate viral retention by ceramic cups closely approximates the 
chemical composition of soil water actually passing the ceramic cups 
installed at the bottom of the large soil cores (see above). As 



■'?'5?IPI^J 



188 



TABLE 5-2. Retention of poliovirus type 1 by porous ceramic cups 



Influent virus 
concentration 

(PFU/ml) 



Effluent virus 
concentration 

(PFU/ml) 



Virus 
retained by 
ceramic cup^ 

(%) . 



2.7 X 10^ 
2.5 X 10^ 



1.8 X 10^ 
1.4 X 10^ 



Mean; 



33.3 
44.0 

38.7 



^Poliovirus was suspended in a rain leachate (conductivity and 
pH equal to 18 ymho/cm at 25°C and 6.0, respectively) from an undis- 
turbed core of Eustis fine sand, and passed through sterile, porous, 
ceramic cups with the use of a vacuum pump at a flow rate of approx. 
2 ml hr~' cm"2. 

Concentration of virus after passage through a porous ceramic 
cup. 

c 
The porous ceramic cups used were 6.9 cm long and had a wall 

thickness of 0.23 cm (no. 2131, Soil Moisture Equipment Corp., Santa 

Barbara, California). The cups used had a pore diameter of 1.4 to 

2.1 pm . 



189 



indicated by the data in Table 5-2, substantial retention of polio- 
virus by the ceramic cups at the bottom of the large soil cores is 
quite likely. In light of the research by Wang et a^. (1980b), a 
significant fraction of echovirus type 1 would probably also be lost 
during passage through the ceramic cups on the large soil cores. 
Due to the possible viral loss, the entire leachate volume from the 
large soil cores must be evaluated for the presence of viruses. 

Application of virus-seeded sludge to soil cores . Sludge 
seeded with poliovirus was applied to two large soil cores (i.e., 
LC3 and LC4~see Figure 5-1) in October 1977, June 1978, and October 
1978. Poliovirus-seeded sludge was also applied to two small soil 
cores (i.e., SCI and SC2— see Figure 5-1) in June 1978 and to one 
small core (i.e., SC2) in October 1978. Sludge seeded with echovirus 
was applied to two other large soil cores (i.e., LCI and LC2— see 
Figure 5-1) and to one small core (i.e., SCI which had received polio- 
virus-seeded sludge in June 1978) in October 1978. Each soil core 
was treated with 2.5 cm of virus-seeded sludge which is equivalent 
to a liquid application rate of 254 m^/ha. The applied sludge was 
allowed to soak in and dry on top of the soil for one to four days. 
During this period, the drying sludge solids on the soil surface of 
the large soil cores were monitored for the presence of viruses, as 
described below. Following the drying period, the sludge resting on 
the soil surface was mixed with the top 2.5 cm of soil. The top 2.5 
cm of soil in the large soil cores was then monitored for the presence 
of seeded viruses as described below. Soil monitoring was continued 



19Q 



until viruses could no longer be detected. The small soil cores were 
not used to study viral survival in sludge-treated soil but rather 
were used to evaluate viral transport as described before. 

Leachates from soil cores . Leachates from all sludge-treated 
soil cores were collected during natural rainfall as shown in Figure 
5-1. In June and July 1978, the small soil cores (i.e., SCI and SC2— 
see Figure 5-1) treated with poliovirus-seeded sludge were periodically 
leached with rain water (see Table 4-2 for chemical characteristics) 
applied from inverted, self-regulated, 1-1 iter Erlenmeyer flasks set 
to maintain a 2.5-cm hydraulic head on the cores (Sanks et al^. 1976). 
The artificial leaching consisted of applying the rain water at a flow 
rate of approximately 3.9 ml/min until approximately one pore volume 
(234 ml for small soil cores) of leachate was collected. All leachate 
samples (i.e., natural or artificial) were promptly taken into the 
laboratory where their volumes were accurately measured [reported volume 
in ml, cm, and pore volumes (calculated according to equations 4-1 
and 4-2)]. The pH and conductivity of each leachate sample were then 
measured using the procedures described above for sludges. Finally, 
each leachate sample was concentrated by membrane filtration and 
assayed for seeded viruses as described below. 

Virus Recovery Procedures 

Sludge . Samples of drying sludge were obtained from the 
surface of the large soil cores (one sample per soil core per sampling 
date). THe solids content of each sludge sample was then determined as 
described below. Seeded viruses (i.e., poliovirus or echovirus) were 



191 



eluted from 1-g (wet weight) samples of drying sludge using the glycine 
procedure developed by Hurst et al_. (1978). This method consisted of 
mixing each 1-g sludge sample with 5 ml of 0.05 M glycine buffer, 
pH 11.5. If necessary, the pH of the mixture was adjusted to between 
10.5 and 11.0 by the addition of IM glycine buffer, pH 11.5. The 
samples were vigorously vortexed for one minute and centrifuged at 
14,000 x g for 5 minutes at 4°C (all centrifugation was performed 
using a Sorvall RC5-B centrifuge, Ivan Sorvall Inc., Norwalk, Con- 
necticut). The supernatants (i.e., the sludge solids eluates) were 
recovered, adjusted to neutral pH by the addition of 1 M glycine buffer, 
pH 2.0, and assayed for eluted viruses as described above. Further 
viral concentration was not required. The entire procedure described 
above was performed in less than 10 minutes. Thus, poliovirus and 
echovirus were subjected to the high pH of 10.5 to 11.0 for no more 
than 10 minutes. Both Hurst et al_. (1978) and Sobsey et al^. (1980) 
observed no appreciable inactivation in 10 minutes of poliovirus type 
1 (LSc) seeded in 0.05 M glycine buffer, pH 10.5 to 11.0. Therefore, 
it is believed that there was no significant inactivation of poliovirus 
or echovirus during the elution of sludge solids. The numbers of 
viruses recovered were expressed as PFU per g dry weight of sludge. 

Soil . Soil samples were obtained from the top 2.5 cm of soil 
in the large soil cores (one sample per soil core per sampling date). 
The moisture content of each soil sample was then determined as 
described below. Seeded viruses (i.e., poliovirus or echovirus) were 
eluted from 10-g (wet weight) samples of soil using the procedure 



192 



described by Bitton et aT_. (1979a). This method consisted of mixing 
each 10-g soil sample with 20 ml of 0.5% (wt./vol.) isoelectric 
casein (Difco Laboratories, Detroit, Michigan),, pH 9.0. If necessary, 
the pH of the mixture was adjusted to between 9.0 and 9.2 by the 
addition of 5 M Trizma base (Sigma Chemical Co., St. Louis, Missouri). 
The samples were vigorously vortexed for 30 seconds and then shaken on 
a rotating shaker for 15 minutes. The samples were subsequently cen- 
trifuged at 1,400 x g for 4 minutes at 4°C. The supernatants (i.e., 
the soil eluates) were recovered and immediately adjusted to neutral 
pH by the addition of 1 M glycine buffer, pH 2.0. Viruses in the soil 
eluates were concentrated by organic flocculation (Katzenelson et al_. 
1976b) as follows. The eluates were adjusted to pH 4.4 by the addition 
of 1 M glycine buffer, pH 2.0. The floe produced were pelleted by 
centrifugation at 160 x g for 1 minute at 4°C. The supernatants were 
discarded. The pellets were mixed with 2 ml of 0.15 M NapHPO,, pH 9.0. 
The mixtures were adjusted to neutral pH by the addition of 1 M glycine 
buffer, pH 11.5, and then magnetically stirred until the pellets were 
completely resolubilized. The samples were subsequently centrifuged 
at 14,000 X g for 10 minutes at 4°C. The supernatants were adjusted 
to neutral pH (i.e., if necessary), adjusted to a final concentration 
of 2% FCS and assaysed for eluted viruses as described above. The 
numbers of viruses recovered were expressed as PFU per g dry weight of 
soil. 

Leachates . Leachate samples were concentrated by membrane 
filtration (Farrah et al. 1976; Hill et ai. 1971; Shuval and Katzenelson 



193 



1972; Sobsey et al. 1973; Sobsey et al. 1980b) as follows. Each 
leachate sample was adjusted to pH 3.5 by the addition of 1 M glycine 
buffer, pH 2.0, and adjusted to a final concentration of 0.0005 M 
aluminum chloride. The treated water was then passed through a series 
of 3.0-, 0.45-, and 0.25-ym Filterite filters in a 47-mm holder. 
Adsorbed viruses were eluted from the filters with 7 ml of PBS con- 
taining 10% FCS, pH 9.0. The filter eluate was adjusted to neutral 
pH by the addition of 1 M glycine buffer, pH 2.0, and assayed for 
seeded viruses as described above. The quantity of poliovirus or 
echovirus detected in each leachate sample was expressed as total PFU 
and as a percentage of the amount of virus applied to the soil (i.e., 
cumulative percent of the total viral PFU applied was calculated 
according to Equation 4-4). 

Measurement of Environmental Parameters 

The soil temperature, soil moisture, and rainfall were 
monitored. The soil temperature was monitored every hour using ther- 
mocouples placed at the soil surface and at depths of 2.5, 10, and 20 
cm on one of the large soil cores as shown on Figure 5-2. The 
thermocouples were connected to an Ester! ine Angus Key Programmable 
Data Acquisition System (Model PD-2064, Esterline Angus Instrument 
Corporation, Indianapolis, Indiana) which printed voltage (millivolts) 
at each thermocouple every hour. The voltages measured were later 
converted to temperature readings with the use of a computer. The 
soil moisture was monitored only when a sample of soil was obtained 
for viral assay. Soil moisture content was determined gravimetrically 



FIGURE 5-2. Photograph of a large soil core of Eustis fine sand 

(LC4) shown with thermocouples placed at the soil sur- 
face and at depths of 2.5, 10, and 20 cm 

The thermocouples were used to monitor the soil 
temperature. Details on the procedures used to 
prepare this soil core appear on pages 183 to 
184 and on pages 193 to 196. 



195 




196 



on a wet-weight basis by drying in an oven at 105°C for 24 hours 

a measured weight of wet soil from the top inch of the soil cores and , 

was expressed as a percentage as follows: 

Soil moisture (%) = wet soil weight (g) - dry soil weight (g) ^ 

' wet soil weight (g) ^ '"" 

(5-1) 

Unfortunately, soil moisture could not be measured more frequently 
because it could not be automated. The rainfall was measured next to 
the soil cores with a farm rain gauge (model no. 510, Science Asso- 
ciates, Inc., Princeton, N.J.) attached to the wooden box as seen in 
Figure 5-1. The rainfall was measured after each rain event in centi- 
meters. Chemical parameters for the rainfall at the experimental site 
are presented in Table 4-2. In the first survival experiment that began 
on 7 October 1977, measurement of some environmental parameters (i.e., 
temperature and rainfall) could not be carried out at the experimental -^ 
site due to the lack of the necessary equipment. In this case, the 
data from the weather station of the Department of Agronomy, University 
of Florida, were used. This station is approximately one mile from the 
experimental site. 

Results and Discussion 
In the previous chapter (i.e.. Chapter IV), virus transport 
through soils was evaluated under controlled laboratory conditions. 
It appeared necessary, however, to study virus transport and survival 
under more natural conditions. Undisturbed soil cores were used to 
assess viral transport and survival under field conditions. In these 



197 



experiments, environmental parameters (i.e., temperature, soil 
moisture, and rainfall) were monitored. The protocol of sludge dis- 
posal to soil was similar to that practiced at sludge disposal sites. 
Virus survival and transport were monitored during three different runs, 
using the same cores. The survival monitoring was terminated when 
viruses were not detectable in soil samples. 

Association between Seeded 
Enteroviruses and Sludge Solids 

Prior to studying virus transport through soil cores, it was 
necessary to assess the extent of virus association with sludge solids. 
Poliovirus was added to aerobically digested sludge and to lagooned 
sludge (2/3 anaerobic and 1/3 aerobic sludge), while echovirus was 
added to lagooned sludge only. Following magnetic stirring for 10 to 
60 minutes, the fraction of sludge solids-associated virus was deter- 
mined. As shown in Table 5-3, more than 90% of poliovirus was found 
associated with sludge solids (aerobic or lagooned sludge). On the 
other hand, only 20.7% of seeded echovirus was observed to be associated 
with lagooned sludge solids (see Table 5-3). The virus-seeded sludge 
was then applied to the undisturbed soils cores. The association 
between viruses and sludge solids may be instrumental in virus retention 
during sludge application to land. 



First Survival Experiment (7 
October 1977-12 October 1977 ) 

During this period, the soil temperature was not monitored. 

However, air temperature data were obtained from the weather station 

of the Department of Agronomy, University of Florida (Figure 5-3). It 



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12 3 4 

Days after sludge application to the soil 



± 



7 October 77 



FIGURE 5-3. 



Daily air temperature (5 ft above ground) for the dura- 
tion of the survival experiment that began 7 October 



Data were collected at the weather station of the 
Department of Agronomy, University of Florida 
Gainesville. This station is approximately I'mile 
from the experimental site (i.e., next to the 
hnyironmental Engineering Sciences building. 
University of Florida, Gainesville). The mean, 
maximum temperature for the period was 30.5°C and 
16 9°c^"' ^^^™^^ temperature for the period was 



200 



is seen that air temperature was as high as 31 °C and as low as 15°C. 
The survival of poliovirus, under natural conditions, following sus- 

Q 

pension in aerobically digested sludge (3.9 x 10 total PFU or 6.3 x 
10^ PFU/g dry weight of sludge) and subsequent application to soil 
cores is shown in Table 5-4. No virus could be recovered in the soil 
samples after three days. The sludge was left on top of the soil for 
three days (i.e., large soil core no. 4— LC4), and mixed thereafter 
with the top 2.5 cm of soil. During that time period, there was more 
than a four log,Q reduction in virus numbers in the drying sludge 
(see LC4— Table 5-4). It is worth noting that during this first 
experiment, the rainfall was low (0.23 cm after five days— see Table 
5-4) and the sludge solids increased from 1.3% to 38%. Dessication 
was probably the major factor which caused the rapid decline of polio- 
virus in the drying sludge and in the soil. 

Second Survival Experiment 
"(2 June 1978-24 AugusFToTS) 

The second survival experiment was initiated in the summer when 
the weather is generally warm and wet in the Gainesville area. Tempera- 
ture data were collected with thermocouples placed at the surface of 
the soil, and at the 2.5-, 10-, and 20-cm depths. Data analysis showed 
that there was no significant difference between soil temperature 
readings at these different depths. Therefore, only the soil 
temperature at the 2.5-cm depth is shown in Figure 5-4. The average 
temperature ranged from 23.5°C to 29°C during a 35-day period beginning 



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FIGURE 5-4. Daily soil temperature (2.5 cm below the soil surface of 
a large core of Eustis fine sand) for the duration of 
the survival experiment that began 2 June 1978 

Data were measured at the experimental site (i.e., 
next to the Environmental Engineering Sciences 
building. University of Florida, Gainesville) 
using a thermocouple placed at the 2.5-cm depth in 
a large (33 cm in length and 15.5 cm internal 
diameter) undisturbed soil core of Eustis fine sand 
(see Table 4-3 ; consists of the Ap and A21 hori- 
zons of this soil). The soil temperature was 
monitored every hour at the 2.5-cm depth, as well 
as at the surface, 10-cm depth, and 20-cm depth. 
From Table 4-3 , it can be seen that all tempera- 
ture readings were made in the Ap horizon of the 
soil. No significant difference was found between 
temperature readings at the surface, 2.5-cm depth, 
10-cm depth, and 20-cm depth. Therefore, only 
the soil temperature at the 2.5-cm depth is 
reported. 



204 



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205 



on 2 June 1978. Figure 5-5 shows the hourly soil temperature profile 
on 17 June 1978. A minimum in soil temperature was observed at 6 a.m. 
and a maximum at 2 p.m. 

With regards to rainfall, the study period was very wet with 
13.63 cm of cumulative rainfall measured from 2 June through 7 July 
1978 (see Table 5-5). Poliovirus survival was monitored in two soil 
cores which had been treated with virus-seeded sludge (see Table 5-5). 
In contrast to the first survival experiment (see Table 5-4), there was 
no drastic decline in virus numbers in the drying sludge prior to the 
sludge being mixed with the top 2.5 cm of soil. Soil monitoring 
revealed that poliovirus could be detected for up to 35 days in both 
soil cores. It is difficult to correlate virus survival with soil 
moisture since this parameter was not continuously monitored. Heavy 
rainfall, however, did not allow the soil (or sludge on the soil sur- 
face) to dry for an extended period of time and this probably con- 
tributed to longer virus survival (see Table 5-5). 

Monitoring of soil leachates from 5 June to 24 August 1978 did 
not reveal any virus, despite their concentration by membrane filtra- 
tion (see Table 5-6). Although 51 cm of rain fell during the study 
period, this represented only 0.5 to 0.7 pore volume. This is the 
reason why we conducted parallel studies with smaller cores (5 cm i.d. 
instead of 15.5 cm i.d.) which were also exposed to natural conditions, 
and treated with virus-seeded sludge and then leached with rainwater 
(the experimental leaching was continued until approximately one pore 
volume of leachate was collected) in addition to natural rainfall. In 
these core studies, some virus breakthrough was observed, but this 



FIGURE 5-5. Hourly soil temperature (2.5 cm below the soil surface 
of a large core of Eustis fine sand) profile for 
17 June 1978 

Soil temperature was measured at the experimental 
site (i.e., next to the Environmental Engineering 
Sciences building. University of Florida, Gaines- 
ville) using a thermocouple placed at the 2.5-cm 
depth in a large soil core of Eustis fine sand 
(LC4— see Figures 5-1 and 5-2). The soil tempera- 
ture was monitored every hour at the 2.5-cm depth, 
as well as at the surface, 10-cm depth, and 20-cm 
depth. From Table 4-3, it can be seen that all 
temperature readings were made in the Ap horizon 
of the soil. No significant difference was found 
between temperature readings at the surface, 2.5- 
cm depth, 10-cm depth, and 20-cm depth. Therefore, 
only the soil temperature at the 2.5-cm depth is 
reported. The average temperature for the day was 
24,2°C. The maximum temperature was 30.8°C and 
it occurred at 2 P.M. The minimum temperature was 
18.9°C and it occurred at 6 A.M. 



207 




12 am 



6 am 12 pm 6 pm 
Time of day on 17 June 78 



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represented only 0.0006% of the total viral input (see Table 5-7). 
These data show that some virus breakthrough can be achieved, under 
saturated flow, in the small cores. This had also been demonstrated 
in laboratory studies (see Figure 4-9). Under unsaturated flow in 

the large cores, however, no such breakthrough occurred (see Table 5-6). 

I 

Third Survival Experiment 

(11 October 1978-20 January 1979) 

From the results of the first two survival experiments, it 
became apparent that with regard to transport pattern, poliovirus type 
1 (LSc) would not be the ideal model virus since it has a high affinity 
for sludge solids (see Table 5-3) and is subsequently immobilized, 
along with the sludge solids, at the top of the soil profile. A virus 
with less affinity for sludge solids would perhaps be more suitable 
for transport studies. Goyal and Gerba (1979) previously found that 
echovirus type 1 (Farouk) poorly adsorbed to soil when compared to 
poliovirus type 1. The association between lagooned sludge solids, 
and poliovirus type 1 (LSc) and echovirus type 1 (Farouk) was, therefore, 
investigated (Table 5-3). As presented above (see Table 5-3), echo- 
virus was less adsorbed (20.7%)to sludge solids than poliovirus 
(95.2%). Lagooned sludge was, thus, seeded with either of these two 
enteroviruses and then applied to soil cores on 11 October 1979. Viral 
presence in the soil and leachates was monitored for 21 and 101 days, 
respectively. 

) During the study period, the average soil temperature, as 
monitored with thermocouples placed in a soil core (LC4--see Figure 5-2), 
ranged from 18°C to 27°C (Figure 5-6). With regard to rainfall, only 



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FIGURE 5-6 Daily soil temperature (2.5 cm below the soil surface of 
a large core of Eustis fine sand) for the duration of 
the survival experiment that began 11 October 1978 

Soil temperature was measured at the experimental 
site (i.e., next to the Environmental Engineering 
Sciences building. University of Florida, Gaines- 
ville) using a thermocouple placed at the 2.5-cm 
depth in a large soil core of Eustis fine sand 
(LC4— see Figures 5-1 and 5-2). The soil tempera- 
ture was monitored every hour at the 2.5-cm depth, 
as well as at the surface, 10-cm depth, and 20-cm 
depth. From Table 4-3, it can be seen that all 
• temperature readings were made in the Ap horizon 
of the soil. No significant difference was found 
between temperature readings at the surface, 2.5- 
cm depth, 10-cm depth, and 20-cm depth. Therefore, 
only the soil temperature at the 2.5-cm depth is 
reported. 



215 










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216 



0.13 cm of rain fell on 11 October 1978 to 1 November 1978 (see Table 
5-8). This was the period during which virus survival was monitored. 
Both poliovirus and echovirus were not detectable in soil after eight 
days of exposure to natural conditions in the dry fall season (see 
Table 5-8). The two enteroviruses were completely inactivated some- 
time between the eighth and the 21st day (see Table 5-8). Soil leach- 
ates were also monitored and a summary of the data is displayed in 
Table 5-9. Neither poliovirus nor echovirus was detected in the 
leachates from all the soil cores (see Table 5-9). 

It appears from these studies that, under conditions prevailing 
in North-Central Florida, enteroviruses are rapidly inactivated during 
sludge application to soils. Their inactivation in the soil appears 
to be affected more by desiccation than by soil temperature. Under 
ideal conditions (warm and dry), a rapid decline of virus was observed 
in the sludge drying on top of the soil and in the top 2.5 cm of soil. 
Other investigators have shown that virus survival in sludge-treated 
soils is prolonged by low temperatures (Damgaard-Larsen et al. 1977; 
Nielsen and Lydholm 1980). Soil leachates collected after natural 
rainfall were negative for both poliovirus and echovirus. Virus 
studies in sludge-amended soils have dealt mainly with the transport 
and survival patterns of enteroviruses and more work is needed on the 
behavior of other enteric viruses, namely rotaviruses, in soils 
receiving wastewater sludges. 



217 



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221 



The results presented above show that the movement of viruses in 
sludge-amended soil cores exposed to natural conditions is limited due 
to the following factors: 



1 



1 



2. 



Viruses were retained with the sludge solids on top 
of the soil profile. 

Viruses were inactivated in the natural environment 
(laboratory leaching studies involving viruses are 
usually completed in one day and neglect to take 
viral inactivation into account - see Figure 4-9). 
Rain water moved through the soil cores under natural, 
unsaturated flow conditions. 
Several investigators have demonstrated that, under natural 
conditions, rain water flows rapidly down macropores causing only 
partial displacement of the initial soil water and contributing greatly 
to groundwater recharge (El rick and French 1966; McMahon and Thomas 
1974; Nielsen and Biggar 1961; Quisenberry and Phillips 1976, 1978; 
Thomas and Phillips 1979; also see review by Burge and Parr 1980). 
Under these conditions, it can be hypothesized that viral particles 
associated with the immobile, matrix, soil water (i.e., in micropores) 
in the sludge-amended cores were probably bypassed by the fast moving 
rain water. In laboratory studies involving saturated flow conditions, 
the reverse would probably occur; that is, the viral particles would 
move rapidly with the soil water in the macropores and this was 
demonstrated in Figure 4-9. 



CHAPTER VI 

MONITORING OF INDIGENOUS ENTEROVIRUSES 

AT TWO SLUDGE DISPOSAL SITES IN FLORIDA 



Introduction 

Wastewater effluents and sludges are being disposed of on land 
with increasing frequency (U.S. Environmental Protection Agency 1974, 
1977, 1978b). It has been suggested, however, that this practice may 
result in the dissemination of viral pathogens throughout the disposal 
site (e.g., soil and groundwater) and adjacent areas (e.g., surface 
waters) (Burge and Marsh 1978; Elliott and Ellis 1977; Foster and 
Engelbrecht 1973; Kowal and Pahren 1978; Pahren 1980} Pahren et al. 
1979). Consequently, numerous studies have been conducted to determine 
the fate of indigenous enteric viruses following wastewater effluent 
application to land at several locations in the United States (Dugan 
etal. 1975; England et al. 1965; Gilbert etal. 1976a, 1976b; Merrell 
and Ward 1968; Schaub and Sorber 1977; Vaughn et al. 1978; Wellings 
et al. 1974, 1975, 1978). In contrast, viral persistence and transport 
at sludge disposal sites has not been adequately investigated (see 
review by Bitton et al. 1979b). Hurst et al. (1978) found that indige- 
nous enteroviruses (mostly echovirus type 7) were inactivated at the 
rate of 2 log-jQ per week in aerobically digested-dewatered sludge 
undergoing further drying in piles on land (temperature range: 20 to 
31°C). These investigators demonstrated that viral inactivation in the 
sludge piles was directly related to the loss of moisture. Viruses 



222 



223 



could not be detected in the sludge after 3 months of drying on the 
soil surface (see Hurst et al_. 1978). When sludge was injected below 
the soil surface, viral persistence was found to be prolonged. At a 
sludge injection site in Butte, Montana, for example, Moore et a^. (1978) 
recovered indigenous enteric viruses (1.1 PFU per gram dry wt. of soil 
obtained from the sludge injection depth--approximately 15 cm) from soil 
sampled 6 months (mostly fall and winter seasons; thus, low temperatures 
were encountered) after sludge injection had been discontinued. Viral 
inactivation was significantly accelerated in injected sludge which 
had seeped to the soil surface at the Butte site and had been subjected 
to air drying (see Moore et al_. 1978). Indigenous enteric viruses at 
sludge disposal sites have been shown not to be transported to surface 
waters (Zenz et al_. 1976) or groundwater (Farrah et al_. 1981a). 

In this chapter, results of viral monitoring at two sludge 
disposal sites in Florida are presented. The City of Gainesville 
(Florida) sludge disposal site adjacent to Lake Kanapaha was monitored 
(monitored the sludge applied to the site, topsoil and groundwater) for 
indigenous enteroviruses on a monthly basis from December 1977 through 
February 1978. Topsoil from the sludge disposal site at the West 
Florida Agricultural Experiment Station, Jay, Florida, was also moni- 
tored for indigenous enteroviruses on a monthly basis from June 1978 
through January 1979. The applied sludge and groundwater at the Jay 
site were monitored for indigenous enteroviruses by Farrah et al . 
(1981a) and their results are sunmarized herein. The information gained 
from viral monitoring at these two sludge disposal sites should be of 



224 



value in the ultimate assessment of the actual viral risk of sludge 
application to soils. 



Materials and Methods 
Sludge Disposal Sites 

Two sludge disposal sites were monitored for indigenous entero- 
viruses and they are described below. 

Kanapaha site . The City of Gainesville (Florida) sludge dis- 
posal site (10 acres or 4.05 ha) adjacent to Lake Kanapaha has been in 
operation since August 1977. At the time of this study, the sludge 
applied to this site originated at the Main Street wastewater treatment 
plant, Gainesville. At this treatment plant, wasted sludge undergoes 
180 days of aerobic digestion. The digested sludge (GDA180--see 
Table 3-2) is conditioned with a cationic polymer and then dewatered 
by centrifugation (U.S. Environmental Protection Agency 1974, 1978a; also 
see Figure 2-1). The conditioned-dewatered sludge was transported by 
tank truck to the Kanapaha site for ultimate disposal. The schematic 
of sludge treatment and final disposal at the Kanapaha site is shown 
in Figure 6-1. The conditioned-dewatered sludge was spread out onto 
the soil and immediately disced into the soil except when a cover crop 
was present. In the presence of a cover crop, the sludge was applied 
as a top dressing on the crop. Coastal bermudagrass was utilized 
during the summer months while ryegrass was used in the winter months 
(Gainesville-Alachua County Regional Utilities 1976). 

The Kanapaha site is depicted in Figure 6-2. As shown, a 60-ft 
(ca. 18-m) deep well in the center (west) of the site was monitored for 



FIGURE 6-1. Scheme for sludge disposal at the Kanapaha site, 
I Gainesville, Florida 



■226 



u. 




<u 




-M 




rC 




CO 
(0 


-o 

S- 

o 


••-> 


U- 


0) 




0) 


<u 


•M 


f ■■ 


to 


^ 


c 


> 




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to 
O 
Q. 
to 



<U 

■o 

=] 
to 

(O 

x: 

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Q. 

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c 
(d 



Wasted sludge 
From the activated sludge unit 


\ f 



Aerobic digestion 
Two digesters in series with total de- 
tention time of approximately 180 days 
(90 days each) 



T 



Sludge conditioning 
Using 1200 mg/il of the cationic polymer, 
Hercofloc #871 (Hercules Co., Atlanta, 
Georgia) 



T 



Sludge dewatering 
By centrifugation at 1400 rpm for 10 
minutes 



T 



Sludge disposal 
Application to 10 acres (4.05 ha) of 
land. Site characteristics: 

1. Soil belongs to Lochloosa series 

2. 1.27 cm/min percolation rate 

3. 50 ft (ca. 15 m) to water table 

4. 128 cm mean annual rainfall 



T 



Sludge application procedure 
The conditioned-dewatered sludge was 
spread on the soil and immediately 
disced into the soil. In the presence 
of a cover crop, the sludge was applied 
— as a ton drP^qin^ nn thP rrnp 





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229 



indigenous enteroviruses. The water table at the site has been found 
to be 50 ft (ca. 15 m) below the soil surface (Gainesville-Alachua 
County Regional Utilities 1976). Other characteristics of the Kanapaha 
site are shown in Figure 6-1. The groundwater flows in a northwesterly 
direction as shown in Figure 6-2. The topsoil at the Kanapaha site was 
also monitored for indigenous enteroviruses (see Figure 6-2). The soil 
found at the site belongs to the Lochloosa series (Gainesville-Alachua 
County Regional Utilities 1976). This soil series is classified as an 
Aquic Arenic Paleudult, loamy, siliceous, hyperthermic (Calhoun et al_. 
1974). Some characteristics of the typifying pedon, Lochloosa fine sand, 
are shown in Table 6-1. 

The conditioned-dewatered sludge, topsoil, and groundwater from 
•this site were monitored for indigenous enteroviruses on a monthly 
basis from December 1977 through February 1978. Wasted sludge and 
aerobically digested sludge (digested 90 days; GDA90 in Table 3-2) from 
the Main Street wastewater treatment plant (Gainesville, Florida) were 
also tested for the presence of indigenous enteroviruses in December 
1977 and February 1978. 

Jay site . Aerobically digested (1/3) and anaerobically digested 
(2/3) sludge from the Montclair and Main Street wastewater treatment 
plants of Pensacola, Florida, respectively, were transported by tank 
truck and discharged into a sludge lagoon located at the West Florida 
Agricultural Experiment Station, Jay, Florida (PDA and PDAN, respec- 
tively; see Table 3-2). Characteristics of the sludge lagoon and the 
scheme for sludge disposal at the Jay site are shown in Figure 6-3. 



230 



TABLE 6-1. Some characteristics of the Lochloosa soil series found 
at the Kanapaha site 



Soiia 
horizon 


Depth 
(cm) 


Mechan 


ical compos 1 


tion (%) 


pH 
(in 1:1 
water) 


Sand 
(2- 
0.05 mm) 


Silt 

(0.05- 

0.002 mm) 


Clay 
(<0.002 mm) 


Ap 


0-18 


88.9 


7.8 


3.3 


4.9 


A21 1 


18-43 


91.2 


4.0 


4.8 


5.1 


A22 


43-71 


89.2 


4.5 


6.3 


5.2 


Bit 


71-81 


79.3 


4.7 


16.0 


5.1 


B21tg 


81-89 


67.4 


4.7 


27.9 


4.8 


B22tg 


89-145 


66.8 


3.6 


29.6 


4.6 


B3g 


145-175 


59.2 


2.6 


38.2 


4.8 



Typifying pedon is Lochloosa fine sand. Data were adapted from 
Calhoun et al. (1974). 



231 



Sludge sources 
Aerobically digested (1/3) and anaerobi 
cally digested (2/3) sludge from the 
Montclair and Main Street wastewater 
treatment plants of Pensacola, Flor- 
ida, respectively. 



Sludge lagoon 
The sludges from Pensacola were held in 
a sludge lagoon at the Jay site. The 
lagoon was approximately 60 by 100 ft 
(ca. 18 by 30 m) and contained approx- 
imately 3.8 X 10° liters of liquid 
sludge. 



T 



Sludge disposal 
The lagooned sludge was applied to 8 
acres (3.24 ha) of land at the Jay 
site. The field was divided into 
plots (see Figure 6-4) which received 
from to 15 acre- inches (0 to 15.4 
ha-cm) of sludge per year. Troup, 
Lucie and Orangeburg soil series 
found at the site. 



T 



Sludge application procedure 
The lagooned sludge was spread on the 
soil, allowed to dry on the soil 
surface for 2 to 14 days and then 
turned under the soil. 



FIGURE 6-3. 



Scheme for sludge disposal at the West Florida Agricul- 
tural Experiment Station, Jay, Florida 



232 



Lagooned sludge (LAG— see Table 3-2) was subsequently spread on 8 
acres (3.24 ha) of land at the Jay site. A diagram of the field used 
for sludge disposal at Jay is shown in Figure 6-4. The field site was 
divided into 72 plots of 40 by 120 ft (ca. 12 by 36 m) which received 
from to 15 acre-inches (0 to 15.4 ha-cm) of sludge per year (see 
Figure 6-4). The applied sludge was allowed to dry on the soil surface 
for 2 to 14 days and was then turned under the soil (see Figure 6-3). 

The soil series found at the site are Troup, Lucie, and Orange- 
burg (see Figure 6-4). The Troup series has been classified as a 
Grossarenic Paleudult, loamy, siliceous, thermic, while the Orangeburg 
series was classified as a Typic Paleudult, fine-loamy, siliceous, 
thermic (Calhoun et al. 1974). Some characteristics of the typifying 
pedons, Troup loamy sand and Orangeburg sandy loam, are shown in 
Table 6-2. No information was available on the Lucie soil series. 
The topsoil from plots numbered 1, 32, and 61 which received 15 acre- 
inches (15.4 ha-cm) of sludge per year was monitored for indigenous 
enteroviruses on a monthly basis from June 1978 through January 1979. 
In addition, the topsoil from the plot numbered 42 which received no 
sludge was monitored for viruses as a control. Indigenous enteroviruses 
in the Pensacola sludges (PDA and PDAN— see Table 3-2) added to the 
sludge lagoon and in the lagooned sludge (LAG— see Table 3-2) were 
monitored by Farrah et ai. (1981a). Also, groundwater from wells on 
the Jay site (see Figure 6-4) was analyzed for the presence of indigenous 
enteroviruses by Farrah etal. (1981a). 



FIGURE 6-4. Diagram of the sludge disposal site at the West Florida 
Agricultural Experiment Station, Jay, Florida 

Lagooned sludge (LAG--see Table 3-2) has been ap- 
plied, for some years, to 8 acres (345,600 ft^ or 
3.24 ha) of land at the West Florida Agricultural 
Experiment Station. As depicted in the figure, 
the disposal site is divided into 72 plots (plot 
number is shown on the top of each plot) of 40 by 
120 ft (ca. 12 by 36 m) which received from to 15 
acre- inches (0 to 15,4 ha-cm) of sludge per year 
(acre-inches applied per year is shown in the 
bottom of each plot). The soil series found at 
the disposal site are Troup, Lucie, and Orangeburg. 
Ttie topsoils in plots numbered 1, 32, 42, and 61 
(designated by triangles) were monitored for the 
presence of indigenous enteroviruses. Groundwater from 
3 wells in the disposal site (designated by 
hexagons) and one well near the sludge lagoon (not 
shown) also was monitored for enteroviruses by 
Farrah et a]_. (1981a). The water table at these 
well sites was 40 to 60 ft (ca. 12 to 18 m) below 
the surface. 



234 



480 ft 



N 



Q. 
O 



Q) 



U 

3 



i- 
3 

<U 
CD 

c 

ro 

S- 

o 



72 


71 


70 


69 


68 


67 


66 


65 


64 


63 


62 


61 

A 


3 


6 


15 





9 


12 





3 


6 


9 


12 


15 


























49 


50 


51 


52 


53 


54 


55 


56 


57 


58 


59 


60 


15 


9 


12 


3 





6 


12 


15 


6 


9 


3 





48 


47 


46 


45 


44 


43 


42 

A/ 


/- 


40 


39 


38 


37 


3 


6 





15 


9 


'^~ 


^ 


3 


6 


9 


12 


15 


25 


26 


27 


28 


29 


#30 


31 


32 

A 


33 


34 


35 


36 


15 


9 


12 


3 


•4 


6 


12 


15 


6 


9 


3 



13 


24 


23 


22 


21 


20 


19 


18 


17 


16 


15 


14 


3 


6 





15 


9 


12 





3 


6 


9 


12 


15 


1 


2 


3 


4 


5 


6 


7 


8 


9 


10 


11 


12 


A 
























15 


9 


12 


3 





6 


12 


15 


6 


9 


3 






o 



A Soils monitored in this study 

# Wells (40-60 ft) monitored by Farrah et al . (1981) 



235 



TABLE 6-2. Some characteristics of the Troup and Orangeburg soil 
series found at the Jay site 



Soil^ 


Soil 
horizon 


Depth 
(cm) 


Mechanical composition {%) 


pH 
(in 1:1 








Sand 

(2- 

0.05 mm) 


Silt 

(0.05- (< 
0.002 mm) 


Clay 
:0.002 mm) 


water) 


Troup 


Ap 


0-15 


79.0 


13.7 


7.3 


6.1 


loamy 
sand 


A21 


15-41 


79.0 


13.2 


7.8 


5.8 




A22 


41-71 


80.0 


12.2 


7.8 


5.4 




A23 


71-91 


80.5 


12.5 


7.0 


5.4 




A24 


91-117 


80.3 


11.9 


7.8 


5.4 




Bit 


117-132 


76.0 


9.6 


14.4 


5.4 




B21t 


132-147 


62.0 


10.0 


28.0 


5.2 


Orangeburg 

sandy 

loam 


Ap 
Bit 


0-20 
20-36 


77.5 
69.6 


13.7 
13.6 


8.8 
16.8 


4.9 
6.1 




B21t 


36-64 


63.5 


12.6 


23.9 


6.3 




B22t 


64-119 


56.9 


8.6 


34.5 


6.3 




B23t 


119-185 


61.8 


8.1 


30.1 


5.6 


^Data 


were adapted from 


Calhoun 


et al. (1974). 







235 



Virus Recovery Procedures 

Sludge . Samples (1 to 4 liters) of wasted sludge, aerobically 
digested sludge (90 days) and dewatered-conditioned sludge from the 
Main Street wastewater treatment plant (Gainesville) were collected 
in sterile Nalgene bottles and transported to the laboratory. The 
pH and solids content of each sludge sample was determined as described 
in Chapter III (seepages 56-59).The sludge samples (i.e., total volume) 
were then centrifuged at 1400 x g for 10 minutes at 4°C. The sludge 
supernatants were discarded. The sludge solids-associated viruses were 
eluted and further concentrated using a modification of the glycine 
method developed by Hurst et^ al_. (1978). This method is detailed in 
Chapter III (see pages 65 to 67). The filter and pellet concentrates 
produced were then assayed for viruses as described below. 

Soil . Composite topsoil samples (200 wet grams) from the 
Kanapaha site were obtained as shown in Figure 6-2. Indigenous entero- 
viruses were recovered from these soil samples using the procedure 
described by Hurst and Gerba (1979). This method consisted of mixing 
each 200-g sample of wet soil with 600 ml of 0.25 M glycine buffer, 
0.05 M ethyl enediaminetetraacetic acid (EDTA), pH 11.5. If necessary, 
the pH of the mixture was adjusted to between 11.0 and 11.5 by the 
addition of 1 M glycine buffer, pH 11.5. The samples were vigorously 
shaken by hand for 30 seconds and then shaken on a rotating shaker for 
4.5 minutes. The samples were subsequently centrifuged at 1400 x g 
for 4 minutes at 4°C (all centrifugation was performed using a Sorvall 
RC5-B centrifuge, Ivan Sorvall Inc., Norwalk, Connecticut). The 



237 



supernatants (i.e., the soil eluates) were recovered and inmediately 
adjusted to neutral pH by the addition of 1 M glycine buffer, pH 2.0. 
The entire procedure described above was performed in approximately 
10 minutes. Viruses in the soil eluates were concentrated by organic 
flocculation (Katzenelson et al^. 1976b) as follows. The eluates were 
adjusted to pH 3.5 by the addition of 1 M glycine buffer, pH 2.0, and 
to 0.06 M aluminum chloride. The floes produced were pelleted by 
centrifugation at 8000 x g for 10 minutes at 4°C. The supernatants 
and pellets produced were treated separately. The supernatants were 
passed through a series of 3.0-, 0.45- and 0.45-vim Filterite filters 
(Filterite Corp., Timonium, Maryland) in a 47-mm holder. Adsorbed 
viruses were eluted from the filters with 7 ml of phosphate-buffered 
saline (PBS) containing 10% fetal calf serum (FCS), pH 9.0. The filter 
eluates were adjusted to neutral pH by the addition of 1 M glycine 
buffer, pH 2.0, and assayed for viruses as described below. The 
pellets previously obtained by centrifuging the samples at pH 3.5 (and 
at 0.06 M aluminum chloride) were mixed with five volumes of PBS 
containing 10% FCS, pH 9.0. The mixtures were adjusted to pH 9.0 by 
the addition of 1 M glycine buffer, pH 11.5, vortexed for 30 seconds and 
then centrifuged at 8000 x g for 10 minutes at 4°C. The supernatants 
were adjusted to neutral pH by the addition of 1 M glycine buffer, pH 
2.0. Viruses in these samples were further concentrated by hydro- 
extraction at 4°C using polyethylene glycol (i.e., Carbowax PEG 20,000 
from Fisher Scientific Co., Pittsburgh, Pennsylvania). The hydroex- 
tracted samples were dialyzed against PBS at 4°C in order to remove 



238 



any possible cytotoxicity and were then assayed for viruses as described 
below. 

Composite topsoil samples (100 wet grams) were obtained from 
the plots shown in Figure 6-4 at the Jay site. Indigenous enteroviruses 
were recovered from these soil samples using the procedure described 
by Bitton et al_. (1979a). This method consisted of mixing each 100-g 
sample of wet soil with 200 ml of 0.5% (wt./vol.) isoelectric casein 
(Difco Laboratories, Detroit, Michigan), pH 9.0. If necessary, the pH 
of the mixture was adjusted to between 9.0 and 9.2 by the addition of 
5 M Trizma base (Sigma Chemical Co., St. Louis, Missouri). The samples 
were vigorously shaken by hand for 30 seconds and then shaken on a 
rotating shaker for 15 minutes. The samples were subsequently centri- 
fuged at 1400 x g for 4 minutes at 4°C. The supernatants (i.e., the 
soil eluates) were recovered and immediately adjusted to neutral pH 
by the addition of 1 M glycine buffer, pH 2.0. Viruses in the soil 
eluates were concentrated by organic flocculation (Katzenelson et al . 
1976b) as follows. The eluates were adjusted to pH 4.4 by the addition 
of 1 M glycine buffer, pH 2,0. The floes produced were pelleted by 
centrifugation at 160 x g for 1 minute at 4°C. The supernatants were 
discarded. The pellets were mixed with 2 ml of 0.15 M Na^HPO^, pH 9.0. 
The mixtures were adjusted to neutral pH by the addition of 1 M glycine 
buffer, pH 11.5, and then magnetically stirred until the pellets were 
completely resolubilized. The samples were subsequently centrifuged 
at 14,000 X g for 10 minutes at 4°C. The supernatants were adjusted to 
neutral pH (i.e., if necessary) and FCS was added to a final concentra- 
tion of 2%. Viruses in these samples were further concentrated by 



239 



ultracentrifugation at 120,000 x g for 2 hours at 5°C in a Tl-60 rotor 
using a Beckman model L3-50 ultracentrifuge (Beckman Instruments, 
Fullerton, California). The pellets produced were suspended in 1 ml of 
FCS. The concentrated samples were sterilized by passage through 
0.25-vim Filterite filters in 13-mm holders and were then assayed for 
viruses as described below. 

Groundwater . Groundwater from a 60-ft (ca. 18-m) well on the 
Kanapaha site (see Figure 6-2) was monitored for indigenous entero- 
viruses. Each groundwater sample of 100 gallons (ca. 384 liters) was 
hand pumped into a 100-gallon tank and was concentrated by membrane fil- 
tration (Fa rr ah eta^, 19.7,6; Hill et al. 1971; Shuval and Katzenelson 
1972; Sobsey et al. 1973; Sobsey et al. 1980b) in the field (see 
Figure 6-5) as follows. The water was adjusted to pH 3.5 by the 
addition of 0.2 N HCl and adjusted to 0.0005 M aluminum chloride. The 
treated water was then passed through a 10-in (ca. 25-cm), 0.25-ym 
pore size Filterite filter. The filter was then treated with 800 ml 
of 0.05 M glycine buffer, pH 11.5. The glycine solution was permitted 
to remain in contact with the filter for 1 minute, was removed and 
then was adjusted to neutral pH by the addition of 1 M glycine buffer, 
pH 2.0. The neutralized sample was transported to the laboratory, and 
within 1 hour, it was adjusted to pH 3.5 by the addition of 1 M glycine 
buffer, pH 2.0, and passed (without prior centrifugation) through a 
series of 3.0- and 0.45-ym Filterite filters in a 47-mm holder. Ad- 
sorbed viruses were eluted from the filters with 7 ml of PBS contain- 
ing 10% FCS, pH 9.0. The filter eluate was adjusted to neutral pH by 





1 






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D1 


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242 



the addition of 1 M glycine buffer, pH 2.0, and assayed for viruses 
as described below. 

Viral Assays 

Samples were serially diluted, if necessary, in PBS containing 
1% FCS (see Appendix for more details on the composition of this 
solution) and then assayed for indigenous enteroviruses on BGM cell 
cultures prepared as described in Chapter III (see pages 53 and 55). 
Inoculated cell cultures were examined for cytopathic effects for 
up to three weeks. Cell cultures showing cytopathic effects were 
passed and viral isolates were titered according to the procedures 
of Farrah et al_. (1981a). The 50% tissue culture infective dose 
(TCID^q) was determined for samples containing indigenous entero- 
viruses. 

Weather Data 

Kanapaha site . Weather data were not available for this sludge 
disposal site. 

Jay site . Weather data were collected at the West Florida 
Agricultural Experiment Station, Jay, Florida, and kindly provided by 
the station's staff. Mean monthly air temperature (maximum and mini- 
mum) and total monthly precipitation from September 1977 through March 
1979 are reported in Figure 6-6. 



243 



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244 



Results and Discussion 
Kanapaha Sludge Disposal Site 

Although indigenous enteroviruses were readily recovered from 
the wasted sludge solids of the Main Street wastewater treatment plant 
(Gainesville, Florida), aerobic digestion for 90 days reduced the 
solids-associated viruses in sludge to almost undetectable levels (see 
Table 6-3). At this treatment plant, the sludge was further digested 
aerobically for an additional 90 days (i.e., total digestion time of 
180 days), conditioned with a cationic polymer, dewatered by centrifu- 
gation, and then disposed of at the Kanapaha site. As shown in Table 
6-3, indigenous enteroviruses could not be recovered from the 
conditioned-dewatered sludge solids. In light of this fact, it is 
understandable that no indigenous enteroviruses were ever detected in 
topsoil and groundwater samples from the Kanapaha site (see Table 6-4). 
Thus, apparently by increasing the sludge digestion time at the waste- 
water treatment plant, the viral hazard of sludge disposal on land was 
eliminated. 

Jay Sludge Disposal Site 

Lagooned sludge (2/3 anaerobic and 1/3 aerobic) has been 
applied to land for many years at the Jay site. The sludge is allowed 
to dry on top of the soil for 2 to 14 days, and then is turned under 
the soil. As shown in Table 6-5, indigenous enteroviruses were not 
detected in 100-g topsoil samples obtained, over an eight-month 
period, from plots at the Jay site which received 15 acre-inches of 
lagooned sludge yearly. Farrah et aj[. (1981a) readily recovered 



245 



TABLE-6-3. Indigenous enteroviruses associated with sludges from 

the Main Street wastewater treatment plant, Gainesville; 
Florida 



Sampling date 



Sludge type 



Viruses detected 
(TCIDgQt^/g dry wt.) 



December 1977 and 
February 1978 

December 1977 and 
February 1978 

December 1977, 
January 1978, and 
February 1978 



Wasted 



Aerobically digested 
(90 days) 

Condi ti oned-dewatered' 



11 to 25 



0.3 to 1.2 



Sludge samples of 1 to 4 liters were centrifuged at 1400 x g 
for 10 min at 4°C. The sludge supernatants produced were discarded. 
The sludge solids were tested for the presence of viruses. 

Refers to the 50% tissue culture infective dose. 

^Identified as sludge GDA90 in Table 3-2. 

Sludge aerobically digested for 180 days (GDA180--see Table 3-2) 
is conditioned with a cationic polymer and then dewatered by centrifu- 
gation at the Main Street plant. The conditioned-dewatered sludge 
was applied to the Kanapaha site (see Table 6-4 for data on the viral 
monitoring of the Kanapaha site). 



246 



TABLE 6-4. Analysis of topsoil and groundwater samples from the 
Kanapaha site for the presence of indigenous entero- 
viruses 



Sampling date 


Cumulative amount of 

sludge^ applied by 

the sampling date 

j'n (cm); 


Sample 


Viruses 
detected 


December 1977 
January 1978 
February 1978 


2.6 (6.5) 

2.6 (6.5) 

3.7 (9.3) 




Topsoil^ 
Groundwater 

Topsoil 
Groundwater 

Topsoil 
Groundwater 













Application of conditioned-dewatered sludge (see Table 6-3) to 
the Kanapaha site began in August 1977. 

Composite topsoil samples (200 wet grams) were obtained monthly 
from the Kanapaha site as shown in Figure 6-2 and were tested for the 
presence of viruses. 

^Groundwater (100 gallons or 384 liters) from a 60- ft (ca. 18-m) 
well on the Kanapaha site (see Figure 6-2) was monitored monthly for the 
presence of viruses. 



247 



TABLE 6-5. Analysis of topsoil samples from the Jay site for the 
presence of indigenous enteroviruses 



Soil 
plot no. 


Amount of lagooned 

sludge° applied yearly 

[acre-in (ha-cm)] 


Total number of 
grams of topsoil 
sampl ed 


Viruses 
detected 


1 


15 (15.4) 


800 





32 


15 (15.4) 


800 





42 





800 





61 


15 (15.4) 


800 






^The locations of these soil plots at the Jay site are shown in 
Figure 6-4. 

The viral content of lagooned sludge is shown in Table 6-6. 

^A composite topsoil sample of 100 wet grams was taken from each 
soil plot monthly from June 1978 through January 1979 and was tested for 
the presence of viruses. 



-'W''^ 



248 



indigenous enteroviruses from the digested sludges added to the lagoon, 
and also from the lagooned sludge (see Table 6-6). However, these 
investigators observed a rapid decline in the number of enteroviruses 
associated with lagooned sludge which had been applied to land. In 
fact, indigenous enteroviruses were almost undetectable in lagooned 
sludge allowed to dry for only two days on the soil surface at the Jay 
site (60% sludge solids content— see Table 6-6). It follows that 
enteroviruses are not likely to be detected in topsoil samples. Thus, 
allowing the sludge to dry on top of the soil before being mixed with 
the soil results in the inactivation of all or most of the viruses 
present (this was also demonstrated using seeded viruses in Chapter V). 
This may be an advantage over sludge injection into soils (Moore et al. 
1978), where viruses can survive for longer periods of time. Despite 
the numerous advantages of sludge injection (aesthetic acceptability, 
and minimal odor and runoff), surface spreading of sludge may result 
in the inactivation of viruses at accelerated rates. It should be 
pointed out that Farrah et al_. (1981a) were unable to detect any 
viruses in groundwater samples obtained from the Jay site (see Table 
6-6). It appears that at this sludge disposal site, as was the case 
at the Kanapaha site described above, enteroviruses pose a minimal 
hazard with respect to soil and groundwater contamination. 



249 



TABLE 6-6- Analysis of sludge and groundwater samples from the Jay 
site for the presence of indigenous enteroviruses 



Sample 



Viruses detected 
(TCIDgQb/g dry wt.) 



Viruses identified 



Digested sludge added 
to the sludge lagoon 

Aerobic 
Anaerobic 

d e 
Lagooned sludge (3%) 



2 to 260 



14 to 260 
2 to 7 

< 0.1 to 100 



Poliovirus 1,2, and 3, 
echovirus 1 and 7, and 
coxsackievirus B4 



Poliovirus 1 and 2, 
echovirus 7 and 15, 
and coxsackievirus 84 



Lagooned sludge applied 
to land 



Day i9%f 
Day 2 (60%)e 
Day 9 (81%)^ 

f 
Groundwater 



<0.01 to 4.6 



1.4 to 4.6 
0.10 to 0.72 
<0.01 to 0.02 



Poliovirus 1, echovirus 
1 , 4, and 7, and 
coxsackievirus 84 



None 



. ^Data were adapted from Farrah et al, (1981a) . 

Refers to the 50% tissue culture infective dose. 

^Aerobically digested and anaerobically digested sludge from the 
Montclair and Main Street wastewater treatment plants of Pensacola, 
Florida, and Pensacola, . Florida, respectively (PDA and PDAN, respec- 
tively; see Table 3-2). Digested sludge samples were obtained from 17 
February 1978 to 12 February 1979. 

Lagooned sludge samples were obtained on a monthly basis from 17 
February 1978 to 24 January 1979. 

^Sludge solids content was expressed as a percentage on a weight 
to volume basis. 

Groundwater from several wells at the Jay site (see Figure 6-4) 
was monitored for the presence of viruses. Over a l.-year period, a 
total of 5,950 liters (1,100 to 2,650 liters per well) of groundwater 
was tested. 



CHAPTER VII 

cnnwTu«. .r ^^^^^'^ ^^ HYDROSTATIC PRESSURE ON THE 
SURVIVAL OF POLIOVIRUS SEEDED IN GROUNDWATER AND SEAWATER 

Introduction 
At several disposal sites in the United States receiving wastewater 
effluent, indigenous enteroviruses have been recovered from groundwater 
(Dugan et al. 1975; Schaub and Sorber 1977; Vaughn et al. 1978; Wellings 
et al. 1974 and 1975). Although indigenous enteroviruses were not 
detected in the groundwater from the two sludge disposal sites described 
in Chapter VI (see pages 244 to 249; also see Farrah et al. 1981a), 
poliovirus type 2 was isolated from a 28-foot (8.5-m) deep well and a 58- 
foot (17.7-m) deep companion well at a sludge disposal site in St. Peters- 
burg, Florida (Wellings et at. 1978). In addition to the'application of 
wastes to land, poor engineering practices (e.g., wells not properly 
sealed and cesspools near wells) have also been demonstrated to result in 
the contamination of groundwater with viral and other pathogens (Allen 
and Geldreich 1975 ; Mack et al. 1972; Robeck 1979). 

It appears, therefore, that viruses sometimes find their way into 
our groundwater supplies. Unfortunately, we know little about the sur- 
vival of viruses in the groundwater environment. In this environment, 
elevated hydrostatic pressures are likely to be encountered (McNabb and 
Dunlap 1975). Such elevated pressures have been found to affect water 
chemistry (Disteche 1959; Hamann 1963; Hamann and Strauss 1955; Home 
and Johnson 1966), and the survival of bacteria (Baross et al. 1975; 
Heden 1964; Horvath and Elkan 1978; Jannasch et al. 1976; 



250 



251 
Morita and ZoBell 1956; Zobell and Cobet 1962; ZoBell and Johnson 1949) and 
tobacco mosaic virus (Johnson et_ al_. 1948; Lauffer and Dow 1941). In 
this chapter, the effect of elevated hydrostatic pressure on the survival 
of poliovirus seeded in groundwater was investigated. For comparative 
purposes, virus survival in seawater under elevated pressure was also 
studied. 

Materials and Methods 
Virus and Viral Assays 

Poliovirus type 1 (strain LSc) was used in the research reported 
in this chapter. Some general properties of polioviruses are shown in 
Table 3-1. Stocks of poliovirus were prepared as described in Chapter 
ni (see page 53 ). Viral stocks were kept at -70°C until used. Polio- 
virus was assayed by the plaque technique as described in Chapter III 
(seepages 53-56). Each viral count shown represents the average of tripli- 
cate counts. The numbers of viruses were expressed as plaque-forming 
units (PFU). 
Water Samples 

The seawater used in this study was sampled at the Mantanzas Inlet 
on Florida's east coast. The groundwater sample was obtained from a 
1200-foot deep well at the Kanapaha wastewater treatment plant, 
Gainesville, Florida. The water samples were collected in sterile 
Nalgene carboys, transported to the University of Florida (Gainesville) 
laboratory and then immediately refrigerated. No chlorine residual was 
found in these water samples (i.e., by the orthotolidine test). The pH 
of each water sample was measured using a digital pH meter model 125 
from Corning (Corning, New York). The conductivity of each vvater : :.' 



252 



sample was measured using a YSI model 33 S-C-T meter (Yellow Springs 
Instrument Co., Inc., Yellow Springs, Ohio). The conductivity and pH 
of each water sample are shown in Table 7-1. The water samples were 
neither filtered nor autoclaved prior to use. 
Poliovirus Exposure to Hydrostatic Pressures 

Poliovirus was added to 75 ml of either seawater or groundwater 
(water sample was temperature acclimated for experimental trial) and 
the solution was mixed for 1 minute. The water sample was then assayed 
in order to determine the initial virus concentration. Following the 
initial viral assay, 65 ml of the virus-seeded water sample was pressur- 
ized (i.e., to between 500 and 4000 psi) directly in a pressure chamber 
(virus-free and temperature acclimated for experimental trial) described 
by Horvath and Elkan (1978). The remaining 10 ml of virus-seeded water was 
left at atmospheric pressure (i.e., 14.7 psi). An atmospheric pressure 
control sample was always run with each elevated pressure trial. The 
elevated and atmospheric pressure water samples were then placed at the 
same temperature (i.e., 2°C or 24°C) for a known period of time (2, 8 and 
24 hours). At the end of the experimental trial, the pressure was 
released, and the water sample was transferred to sterile glassware 
and assayed for poliovirus. The atmospheric pressure water sample was 
also assayed for poliovirus. Two experimental trials were run at each 
elevated pressure condition tested. Poliovirus recovery after exposure 
to elevated pressure was expressed as a percentage of the viral titer 
in the atmospheric pressure sample. 



253 



TABLE 7-1. Conductivity and pH of water samples used in this study 



Water sample 



Conductivity 
(ymho/cm at 25°C) 



pH 



Seawater 
Groundwater 



40,000 
475 



7.7 
7.9 



254 



Results and Discussion 
Initially, it was important to determine the effect of elevated 
pressure on the temperature and pH of groundwater. As shown in Table 7-2, 
the pH and temperature of groundwater was not markedly changed following 
pressurization at 3000 psi for 24 hours. 

The inactivation of poliovirus in seawater subjected to 1000 psi 
of hydrostatic pressure was found to increase as the pressurization time 
was increased from 2 to 24 hours (see Table 7-3). After 24 hours of 
exposure to 1000 psi of hydrostatic pressure, only 15.6% of seeded polio- 
virus was recovered. Clearly, poliovirus in seawater was inactivated at 
an accelerated rate under 1000 psi of pressure relative to the control at 
atmospheric pressure. No such inactivation was observed for poliovirus 
in groundwater even after exposure to as high as 4000 psi of hydrostatic 
pressure (see Table 7-4). Thus, hydrostatic pressures in groundwater are 
not likely to increase viral inactivation. 

High hydrostatic pressures have been previously shown to inacti- 
vate bacterial enzyme systems (Morita 1967; Morita and ZoBell 1956), to 
retard the growth of mesophilic terrestrial bacterie (Horvath and Elkan 
1978; ZoBell and Cobet 1962; ZoBell and Johnson 1949), and to accelerate 
the death rate of mesophilic bacteria (Baross et al. 1975; Morita 1967; 
ZoBell and Cobet 1962). Although tobacco mosaic virus (Lauffer and Dow 
1941), and 1^ and T^ bacteriophages (Heden 1964) have been demonstrated 
to be inactivated at pressures in excess of 1900 atm, little work had 
previously been done on the effect of hydrostatic pressure on animal 
viruses. The research presented above is only preliminary and more work 
is needed on the effect of hydrostatic pressure on viruses. 



255 



TABLE 7-2. Effect of hydrostatic pressure on the temperature and pH 
of groundwater 



Pressure^ Mean Mean pH 



(psi) temperature 

(°C) 



14.7 24 8.1 

(atmospheric) 



3,000 25 8.3 



^Pressurization time was 24 hours. 



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CHAPTER VIII 
CONCLUSIONS 

Based on the findings of this study, the following conclusions can 
be drawn: 

1. Poliovirus type 1 (LSc) was largely associated with digested, 
conditioned-dewatered, chemical (alum, ferric chloride and lime) 
and lime-stabilized, chemical sludge (alum and ferric chloride) 
solids. 

2. Sludge type was found to affect the degree of association between 
seeded poliovirus and sludge solids. The mean percent of solids- 
associated viruses for activated sludge mixed liquors, anaerobic- 
ally digested sludges and aerobically digested sludges was 57, 

70 and 95, respectively. The degree of association between polio- 
virus and sludge solids was significantly greater for aerobically 
digested sludges than for the other two sludge types. 

3. A smaller fraction of echovirus type 1 (Farouk) was associated 
with lagooned sludge solids than was the case for poliovirus. 

4. The effectiveness of the glycine method in the recovery of solids- 
associated viruses was found to be affected by sludge type. Sig- 
nificantly lower mean poliovirus recovery was found for aerobic- 
ally digested sludges (15%) than for mixed liquors or anaerobic- 
ally digested sludges (72% and 60%, respectively). 

5. Poliovirus transport studies involving soil cores treated with 
virus-seeded sludge were conducted under controlled laboratory 
and saturated flow conditions. A Red Bay sandy loam displayed 
a substantially greater retention capacity for poliovirus in 
anaerobically digested sludge than a sandy soil (i.e., Eustis 
fine sand). The Red Bay sandy loam soil was shown to completely 
retain poliovirus following the application of conditioned- 
dewatered, chemical (alum, ferric chloride and lime) and lime- 
stabilized, chemical (alum and ferric chloride) sludge. 

258 



259 



6. The stabilization of chemical sludges (alum and ferric chloride 
sludges) with lime resulted in almost complete inactivation of 
seeded poliovirus. 

7. Undisturbed soil cores of Eustis fine sand were treated with 
several inches of virus-seeded (poliovirus and echovirus) sludge 
during a two-year period. The soil cores were exposed to natural 
conditions and soil temperature, soil moisture and rainfall were 
monitored. Both viruses were found to be rapidly inactivated in 
the sludge during the drying process on top of the soil cores. 
Monitoring of the top inch of soil revealed that both viruses 
were inactivated with time and were undetectable after 35 days. 
The inactivation of both viruses in the soil appears to be affect- 
ed more by soil moisture. Soil leachates collected after natural 
rainfall (unsaturated flow conditions) were negative for both 
viruses except on one occasion (only 0.0006% of total poliovirus 
applied was found in leachate) when heavy rainfall occurred 
immediately after liquid sludge application to the soil. 

8. Indigenous enteroviruses were not detected in topsoil and ground- 
water samples from two sludge disposal sites in Florida. It 
appears that, at these two sludge disposal sites, enteroviruses 

. pose a minimal hazard with respect to soil and groundwater 

contamination. 

9. Poliovirus in seawater was found to be inactivated when subjected 
to 1000 psi of hydrostatic pressure for 24 hours. No such in- 
activation was observed for this virus in groundwater even after 
exposure to as high as 4000 psi of hydrostatic pressure. 

This research has allowed the determination of the persistence and possible 
movement of pathogenic viruses in soils treated with wastewater sludge. 
The information gained from this study is of value in the ultimate assess- 
ment of the potential risk of viral infection to humans associated with 
land disposal of sludges. 



APPENDIX 

COMPOSITION OF MEDIA AND SOLUTIONS 

USED IN ENTEROVIRUS ASSAYS 

1. Gey's Balanced Salt Solution (BSS) is the common diluent for cell 
cultures: 

Gey's A (lOx): 70 grams NaCl 

3.7 grams KCl 

3.01 grams Na2HP04 • I2H2O 
0.237 grams KH2PO4 
100 ml 0.1% phenol red 
10 grams glucose 
900 ml glass distilled water 
5 ml chloroform, as a preservative 

This stock solution of Gey's A is stored at room 
temperature unautoclaved, and is diluted 1:10 and 
autoclaved when needed. 

Gey's B (20x) : 0.42 grams MgCl2 • 6H2O 

0.14 grams MgS04 * 7H20 
0.34 grams CaCl2 
100 ml glass distilled water 

Gey's C (20x): 2.25 grams NaHCOs 

100 ml glass distilled water 

Bubble CO2 into Gey's C until pH is less than 7.6. 
Dispense and tightly cap. 

Gey's B and C are autoclaved without further dilution. 

To make the complete Gey's Balanced Salt Solution (BSS) add: 

90 parts Gey's A (Ix) 
5 parts Gey's B (20x) 
5 parts Gey's C (20x) 

2. Hepes buffer (1 M) stock solution: 

47.7 grams Hepes 
190 ml Gey's A (Ix) 



260 



261 



10 ml Gey's B (20x) 

16 ml 2 M NaOH (8g/100 ml) 



Dispense and autoclave. 



3. Streptomycin-penicillin (lOOOx) stock solution: 

Solution I: 1.0 gram streptomycin 

8 ml Gey's A (Ix) 

Solution II: 10^ units of penicillin 

4 ml of Solution I 

Solution II contains 125 mg of streptomycin and 
2.5 X 105 units of penicillin per ml which is 
lOOOx of what is required. Therefore, it must 
be diluted 1:1000 in the final solution. 



4. Eagle's Minimal Essential Medium (MEM) using Gey's BSS plus 10% 
fetal calf serum (PCS) (i.e., growth medium): 

300 ml Gey's A (Ix) 
20 ml Gey's 8 (20x) 
20 ml Gey's C (20x) 
8 ml MEM essential amino acids (50x) 

(International Scientific, Gary, 

Illinois) 
4 ml vitamins (lOOx) (International 

Sci .) 

4 ml glutamine (lOOx) (International 

Sci .) 
0.4 ml streptomycin-penicillin stock 

(lOOOx) 
40 ml PCS (International Sci.) 

5. Solutions required for the removal of cells from glass (trypsini- 
zation): 

Solution I (pre-trypsin wash): 

300 ml Gey's A (Ix) 

5 ml Gey's C (20x) 

Dispense and autoclave. This solution removes 
all traces of serum (which contains trypsin inhibi- 
tors) as well as Ca+2 and Mg+2 ions. 



262 



; Solution II [1% versene (i.e., EDTA) stock in Gey's A]: 

2.0 grams ethylenediamine- 
tetraacetic acid (EDTA) 
10 ml 2 M NaOH (8g/100 ml) 
20 ml Gey's A (lOx) 
170 ml glass distilled water 

Dispense and autoclave. 

Solution III (2.5% trypsin stock): 

1.0 gram trypsin (Difco Laboratories, 

Detroit, Michigan; 1:250) 
100 ml glass distilled water 

Sterilize by cold filtration. Dispense in 5 ml 
aliquots to screw-capped test tubes and freeze for 
storage. 

Solution IV (standard trypsin-versene solution): 

100 ml Gey's A (Ix) 
I ■ 4 ml Gey's C (20x) 

4 ml stock trypsin (2.5%) 
[' 4 ml stock versene (1% in Gey's A) 

This solution is good for only one day. This 
solution is used to remove the cells from the 
32-ounce bottles in which they have been growing 
prior to their distribution to plaque bottles. 

6. Methyl cellulose overlay for cell cultures (1% methyl cellulose 
plus 5% PCS): 

Solution I: 300 ml glass distilled water 

6 grams methyl cellulose 
(1500 centi poise) 

[ Autoclave and then allow to cool to room temperature. 



shaking vigorously every hour to avoid layering, 
Refrigerate. 

Solution II (2x Eagle's MEM): 

350 ml glass distilled water 
120 ml Eagle's MEM (lOx) with Hanks' 
salts (International Sci.) 



263 



50 ml Gey's C (20x) 

60 ml FCS (International Sci.) 

25 ml Hepes buffer (1 M) stock 

solution 
12 ml glutamine (lOOx) (International 

Sci . ) 
1.2 ml streptomycin-penicillin stock 

(lOOOx) 



Combine equal amounts of Solutions I and II to make the methyl 
cellulose overlay. To 500 ml of the methyl cellulose overlay, 
0.175 ml of kanamycin stock (i.e., stock supplied as a liquid at a 
concentration of 1 gram per 3 ml). 



7. Crystal violet: 

Solution I: 20 grams crystal violet 

200 ml absolute ethanol 

Allow this solution to sit overnight. 

Solution II: 8 grams amnonium oxalate 

800 ml distilled water 

Mix Solutions I and II, and dilute 1:10 with tap water. This 
stain is used to make the plaques on the cell monolayer visible 
to the naked eye. In some experiments, the cells were stained, 
instead, with 0.5 ml of 0.5% neutral red. 



8. Eagle's MEM using Gey's BSS plus 5% calf serum and 0.03 M Hepes 
buffer at pH 7: 

This solution was used to make some virus dilu- 
tions. The solution is made by substituting, in 
Solution 4 above, 20 ml of calf serum (International 
Sci.), 12 ml of 1 M Hepes buffer stock solution 
and 8 ml of Gey's A (Ix) for 40 ml of fetal calf 
serum. 



9. Phosphate-buffered saline (PBS) at pH 7.4-7.6: 



8.0 grams NaCl 
0.2 grams KCl 
1.15 grams Na2HP04 



264 



0.2 grams KH2PO4 

1000 ml deionized water 



Autoclave. 



10. PBS. containing 2% fetal calf serum at pH 7.4 (this solution, was 
also used to make virus dilutions.); 

To 490 ml of PBS, add the following aseptically: 

10 ml fetal calf serum (International 

Sci .) 
0.5 ml phenol red stock (0.5%) 
0.5 ml streptomycin-penicillin stock 

(lOOOx) 



11. NaCT. stock (used to bring an undiluted sample to isotonicity--i.e. , 
0.85 grams NaCl per 100 ml): 

17.0 grams NaCl 

100 ml deionized water 

Autoclave. Dilute 1/20 in final sample. 



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BIOGRAPHICAL SKETCH 

Oscar Carlos Pancorbo was born November 27, 1953, in Mantanzas, 
Cuba. There, he attended parochial school until his immigration to 
the United States in October 1961. In May 1971, he graduated from 
Immaculata-La Salle High School in Miami, Florida. In August 1974, 
he graduated from the University of Florida with a Bachelor of Science 
in zoology with Honors. Following graduation, he enrolled in the 
Graduate School of the University of Florida and in June 1976, was 
awarded the degree of Master of Science in environmental engineering 
Sciences. Since August 1981 , he has been employed as an Assistant 
Professor in the Department of Environmental Health at East Tennessee 
State University, Johnson City. 

Oscar Carlos Pancorbo is a member of the American Society for 
Microbiology, American Association for the Advancement of Science, 
Florida Academy of Sciences, and of the Honor Societies of Phi Kappa 
Phi (General Scholarship), Tau Beta Pi (Engineering) and Epsilon Nu 
Eta (Environmental Health). He is married to the former Ambrosina 
Pita and they have two daughters, Adrianne and Amanda. 



I certify that I have read this study and that in my opinion 
it conforms to acceptable standards of scholarly presentation and 
is fully adequate, in scope and quality, as a dissertation for the 
degree of Doctor of Philosophy. 





Bitton, LTiairman 
sor of Environmental Engineering Sciences 



I certify that I have read this study and that in my opinion 
if conforms to acceptable standards of scholarly presentation and 
is fully adequate, in scope and quality, as a dissertation for the 
degree of Doctor of Philosophy. 




(ias'L. Crisman' 

Associate Professor of Environmental Engineering 
Sciences 



I certify that I have read this study and that in my opinion 
it conforms to acceptable standards of scholarly presentation and 
is fully adequate, in scope and quality, as a dissertation for the 
degree of Doctor of Philosoohy. 



Dale A. Lundgren T / 



.undgrer 
Professor of Environmental 



Engineering Sciences 



I certify that I have read this study and that in my opinion 
it conforms to acceptable standards of scholarly presentation and 
is fully adequate, in scope and quality, as a dissertation for the 
degree of Doctor of Philosophy. 



A. r^7/.y 



George E. Gifford 

Professor of Immunology and Medical Microbiology 



I certify that I have read this study and that in my opinion 
it conforms to acceptable standards of scholarly presentation and 
is fully adequate, in scope and quality, as a dissertation for the 
degree of Doctor of Philosophy, 



Samuel R. Farrah 



Associate Professor of Microbiology and 
Cell Science 



I certify that I have read this study and that in my opinion 
it conforms to acceptable standards of scholarly presentation and 
is fully adequate, in scope and quality, as a dissertation for the 
degree of Doctor of Philosophy, 



Allen R. Overman 



Professor of Agricultural Engineering 



This dissertation was submitted to the Graduate Faculty of the College 
of Engineering and to the Graduate Council, and was accepted as partial 
fulfillment of the requirements for the degree of Doctor of Philosophy. 



August, 1982 I'UJUJU. UM^^ 

Dean, College of Engineering 



iLluidk 



Dean for Graduate Studies and Research 



UNIVERSITY OF FLORIDA 



3 1262 08554 8476